Methods for displaying (poly) peptides/proteins on bacteriophage particles via disulfide bonds

ABSTRACT

The present invention relates to methods for displaying (poly)peptides/proteins on the surface of bacteriophage particles by attaching the (poly)peptide/proteins via disulfide bonds.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is based upon, and claims priority to, European patentapplications EP 99 11 4072.4 and EP 00 103551.8 and PCT applicationPCT/EP00/06968, filed Jul. 20, 2000, which are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

The present invention relates to methods for displaying(poly)peptides/proteins on the surface of bacteriophage particles byattaching the (poly)peptide/proteins via disulfide bonds. A number ofdocuments are cited throughout this specification. The disclosurecontent of these documents is herewith incorporated by reference intheir entirety.

Smith first demonstrated in 1985 that filamentous phage tolerate foreignprotein fragments inserted in their gene III protein (pIII), and couldshow that the protein fragments are presented on the phage surface(Smith, 1985). Ladner extended that concept to the screening ofrepertoires of (poly)peptides and/or proteins displayed on the surfaceof phage (WO 88/06630; WO 90/02809) and, since then, phage display hasexperienced a dramatic progress and resulted in substantialachievements.

Various formats have been developed to construct and screen(poly)peptide/protein phage-display libraries, and a large number ofreview articles and monographs cover and summarise these developments(e.g., Kay et al., 1996; Dunn, 1996; McGregor, 1996).

Most often, filamentous phage-based systems have been used.

Initially proposed as display of single-chain Fv (scFv) fragments (WO88/06630; see additionally WO 92/01047), the method has rapidly beenexpanded to the display of bovine pancreatic trypsin inhibitor (BPTI)(WO 90/02809), peptide libraries (WO 91/19818), human growth hormone (WO92/09690), and of various other proteins including the display ofmultimeric proteins such as Fab fragments (WO 91/17271; WO 92/01047).

To anchor the peptide or protein to the filamentous bacteriophagesurface, mostly genetic fusions to phage coat proteins are employed.Preferred are fusions to gene III protein (Parmley & Smith, 1988) orfragments thereof (Bass et al., 1990), and gene VIII protein (Greenwoodet al., 1991). In one case, gene VI has been used (Jespers et al.,1995), and recently, a combination of gene VII and gene IX has been usedfor the display of Fv fragments (Gao et al., 1999).

Furthermore, phage display has also been achieved on phage lambda. Inthat case, gene V protein (Maruyama et al., 1994), gene J protein, andgene D protein (Sternberg & Hoess, 1995; Mikawa et al., 1996) have beenused.

Besides using genetic fusions, foreign peptides or proteins have beenattached to phage surfaces via association domains. In WO 91/17271, itwas suggested to use a tag displayed on phage and a tag binding ligandfused to the peptide/protein to be displayed to achieve a non-covalentdisplay.

A similar concept was pursued for the display of cDNA libraries (Crameri& Suter, 1993). There the jun/fos interaction was used to mediate thedisplay of cDNA fragments. In their construct, additional cysteineresidues flanking both ends of jun as well as fos further stabilised theinteraction by forming two disulfide bonds

When screening phage display libraries in biopanning the problem remainshow best to recover phage which have bound to the desired target.Normally, this is achieved by elution with appropriate buffers, eitherby using a pH- or salt gradient, or by specific elution using solubletarget. However, the most interesting binders which bind with highaffinity to the target might be lost by that approach. Severalalternative methods have been devised which try to overcome thatproblem, either by providing a cleavage signal between the(poly)peptide/protein being displayed and its fusion partner, or betweenthe target of interest and its carrier which anchors the target to asolid surface.

Furthermore, all the approaches referred to hereinabove require to usefusion proteins comprising at least part of a phage coat protein and aforeign (poly)peptide/protein. Especially in the case of using gene IIIas partner for peptides/proteins to be displayed, this leads to severalproblems. First, the expression product of gene III is toxic to the hostcell, which requires tight regulation of gene III fusion proteins.Second, expression of gene III products can make host cells resistant toinfection with helper phage required for the production of progeny phageparticles. And finally, recombination events between gene III fusionconstructs and wild type copies of gene III lead to undesired artefacts.Furthermore, since at least the C-terminal domain of the gene IIIprotein comprising about 190 amino acids has to be used in order toachieve incorporation of the fusion protein into the phage coat, thesize of the vectors comprising the nucleic acid sequences is ratherlarger, leading to a decrease in transformation efficiency.Transformation efficiency, however, is a crucial factor for theproduction of very large libraries. Additionally, for thecharacterisation of (poly)peptide/proteins obtained after selection froma phage display library, the (poly)peptide/protein are usually reclonedinto expression vectors in order to remove the phage coat protein fusionpartner, or in order to create new fusion proteins such as by fusion toenzymes for detection or to multimerisation domains. It would beadvantageously to have a system which would allow direct expressionwithout recloning, and direct coupling of the (poly)peptide/protein toother moieties.

Furthermore, most of these approaches (except for the work of Jespers etal. (1995), WO 91/17271, and Crameri & Suter (1993) mentionedhereinabove) are limited to the presentation of (poly)peptides/proteinshaving a free N-terminus, since the (poly)peptides/proteins have to befused at the C-terminus with a phage coat protein. Especially in thecase of cDNA libraries, or in the case of proteins requiring a freeC-terminus to be functional, it would be highly desirable to have asimple method which doesn't require the generation of C-terminalfusions.

SUMMARY OF THE INVENTION

Thus, the technical problem underlying the present invention is todevelop a simple, reliable system which enables the presentation of(poly)peptides/proteins on phage particles without the need to usefusion proteins with phage coat proteins. Additionally, there is a needfor a method which allows to recover tightly binding(poly)peptides/proteins in a more reliable way.

The solution to this technical problem is achieved by providing theembodiments characterised in the claims. Accordingly, the presentinvention allows to easily create and screen large libraries of(poly)peptides/proteins displayed on the surface of bacteriophageparticles. The technical approach of the present invention, i.e. linking(poly)peptides/proteins by disulfide bonds to the surface of phageparticles, is neither provided nor suggested by the prior art.

Thus, the present invention relates to a method for displaying a(poly)peptide/protein on the surface of a bacteriophage particlecomprising:

causing or allowing the attachment of said (poly)peptide/protein afterexpression to a member of the protein coat of said bacteriophageparticle, wherein said attachment is caused by the formation of adisulfide bond between a first cysteine residue comprised in said(poly)peptide/protein and a second cysteine residue comprised in saidmember of the protein coat.

DETAILED DESCRIPTION OF THE INVENTION

In the context of the present invention, the term “bacteriophage”relates to bacterial viruses forming packages consisting of a proteincoat containing nucleic acid required for the replication of the phages.The nucleic acid may be DNA or RNA, either double or single stranded,linear or circular. Bacteriophage such as phage lambda or filamentousphage (such as M13, fd, or fl) are well known to the artisan of ordinaryskill in the art. In the context of the present invention, the term“bacteriophage particles” refers to the particles according to thepresent invention, i.e. to particles displaying a (poly)peptide/proteinvia a disulfide bonds. During the assembly of bacteriophages, the coatproteins may package different nucleic acid sequences, provided thatthey comprise a packaging signal. In the context of the presentinvention, the term “nucleic acid sequences” contained in bacteriophagesor bacteriophage particles relates to nucleic acid sequences or vectorshaving the ability to be packaged by bacteriophage coat proteins duringassembly of bacteriophages or bacteriophage particles. Preferably saidnucleic acid sequences or vectors are derived from naturally occurringgenomes of bacteriophage, and comprise for example, in the case offilamentous phage, phage and phagemid vectors. The latter are plasmidscontaining a packaging signal and a phage origin of replication inaddition to plasmid features.

The term “(poly)peptide” relates to molecules consisting of one or morechains of multiple, i.e. two or more, amino acids linked via peptidebonds.

The term “protein” refers to (poly)peptides where at least part of the(poly)peptide has or is able to acquire a defined three-dimensionalarrangement by forming secondary, tertiary, or quaternary structureswithin and/or between its (poly)peptide chain(s). This definitioncomprises proteins such as naturally occurring or at least partiallyartificial proteins, as well as fragments or domains of whole proteins,as long as these fragments or domains are able to acquire a definedthree-dimensional arrangement as described above.

Examples of (poly)peptides/proteins consisting of one chain aresingle-chain Fv antibody fragments, and examples for(poly)peptides/proteins consisting of more chains are Fab antibodyfragments.

When the first cysteine residue is located at the C-terminus of the(poly)peptide/protein, the display format corresponds to theconventional display set-up with the C-terminus being genetically fusedto the member of the phage coat protein. However, by using theN-terminus of the (poly)peptide/protein, the display format can bereverted as in the pJuFO system of Crameri & Suter referred to above.

The term “surface of a bacteriophage particle” refers to the part of abacteriophage particle which is in contact with the medium the particleis contained in and which is accessible. The surface is determined bythe proteins being part of the phage coat (the members of the proteincoat of the particle) which is assembled during phage production inappropriate host cells.

The term “after expression” refers to the situation that nucleic acidencoding said (poly)peptide/protein is expressed in a host cell prior toattachment of the (poly)peptide/protein to said coat, in contrast toapproaches where nucleic acid encoding fusion proteins withbacteriophage coat proteins are being expressed. The expression ofnucleic acid encoding said (poly)peptide/protein and the step of causingor allowing the attachment may be performed in separated steps and/orenvironments. Preferably, however, expression and the step of causing orallowing the attachment are being performed sequentially in anappropriate host cell. The term “wherein said attachment is caused bythe formation of a disulfide bond” refers to a situation, wherein thedisulfide bond is responsible for the attachment, and wherein nointeraction domain for interaction with a second domain present in the(poly)peptide/protein has been recombinantly fused to said member of theprotein coat, as for example in the case of the pJuFo system (Crameri &Suter, 1993).

In a preferred embodiment, the bacteriophage particle displaying the(poly)peptide/protein contains a nucleic acid sequence encoding the(poly)peptide/protein.

Methods for construction of nucleic acid molecules encoding a(poly)peptide/protein according to the present invention, forconstruction of vectors comprising said nucleic acid molecules, forintroduction of said vectors into appropriately chosen host cells, forcausing or allowing the expression of said (poly)peptides/proteins arewell-known in the art (see, e.g., Sambrook et al., 1989; Ausubel et al.,1999; Ge et al, 1995). Further well-known are methods for theintroduction of genetic material required for the generation of progenybacteriophages or bacteriophage particles in appropriate host cells, andfor causing or allowing the generation of said progeny bacteriophages orbacteriophage particles (see, e.g., Kay et al., 1996).

In a further preferred embodiment, the present invention relates to amethod, wherein said second cysteine residue is present at acorresponding amino acid position in a wild type coat protein of abacteriophage.

In a yet further preferred embodiment, the present invention relates toa method, wherein said member of the protein coat is a wild type coatprotein of a bacteriophage.

The term “wild type coat protein” refers to those proteins forming thephage coat of naturally occurring bacteriophages. In the case offilamentous bacteriophage, said wild type proteins are gene III protein(pIII), gene VI protein (pVI), gene VII protein (pVII), gene VIIIprotein (pVIII), and gene IX protein (pIX). The sequences, including thedifferences between the closely related members of the filamentousbacteriophages such as f1, fd, and M13, are well known to one ofordinary skill in the art (see, e.g., Kay et al., 1996).

In a further preferred embodiment, said member of the protein coat is atruncated variant of a wild type coat protein of a bacteriophage,wherein said truncated variant comprises at least that part of said wildtype coat protein causing the incorporation of said coat protein intothe protein coat of the bacteriophage particle.

The term “truncated variant” refers to proteins derived from the wildtype proteins referred to above which are modified by deletion of atleast part of the wild type sequences. This comprises variants such astruncated gene III protein variants which have been found inbacteriophage mutants (Crissman & Smith, 1984) or which have beengenerated in the course of standard phage display methods (e.g. Bass etal., 1990; Krebber, 1996). For example, said truncated variant mayconsist, or include, the C-terminal domain of the gene III protein. Toidentify truncated variants according to the present invention, adetection tag may be fused to the variant, and an assay may be set up todetermine whether the variant is incorporated into the phage coat ofbacteriophage particles formed in the presence of the variant. By way oftruncating a wild type protein by deleting a part of the wild typeprotein, a cysteine residue may become available which in the wild typeprotein was forming a disulfide bond with a second cysteine comprised inthe deleted part.

In a yet further preferred embodiment, said member of the protein coatis a modified variant of a wild type coat protein of a bacteriophage,wherein said modified variant is capable of being incorporated into theprotein coat of the bacteriophage particle.

Methods for achieving modification of a wild type protein according tothe present invention are well-known to one of ordinary skill in theart, and involve standard cloning and/or mutagenesis techniques. Methodsfor the construction of nucleic acid molecules encoding a modifiedvariant of a wild type protein used in a method according to the presentinvention, for construction of vectors comprising said nucleic acidmolecules, including the construction of phage and/or phagemid vectors,for introduction of said vectors into appropriately chosen host cells,for causing or allowing the expression of said modified protein arewell-known in the art (see, e.g., Sambrook et al., 1989; Ausubel et al.,1999; Kay et al., 1996). To identify modified variants according to thepresent invention, a detection tag may be fused to the variant, and anassay may be set up to determine whether the variant is capable or beingincorporated into the phage coat of bacteriophage particles formed inthe presence of the variant.

In a most preferred embodiment, said second cysteine residue is notpresent at a corresponding amino acid position in a wild type coatprotein of a bacteriophage.

In a preferred embodiment, said second cysteine has been artificiallyintroduced into a wild type coat protein of a bacteriophage.

In the context of the present invention, the term “artificiallyintroduced” refers to a situation where a wild type coat protein hasbeen modified by e.g. recombinant means. For example, nucleic acidencoding a wild type coat protein may be manipulated by standardprocedures to introduce a cysteine codon creating a nucleic acidsequence encoding a modified coat protein, wherein a cysteine residue isartificially introduced by insertion into, or addition of said cysteineresidue to, said at least part of a wild type or modified coat protein,or by substitution of an amino acid residue comprised in said at leastpart of a wild type or modified protein by said cysteine residue, or byfusion of said at least part of a wild type or modified coat proteinwith a (poly)peptide/protein comprising said second cysteine residue, orby any combination of said insertions, additions, substitutions orfusions. Upon expression of the nucleic acid comprising suchrecombinantly introduced cysteine codon, a variant of the wild typeprotein is formed comprising a cysteine residue.

In a further most preferred embodiment, said second cysteine has beenartificially introduced into a truncated variant of a wild type coatprotein of a bacteriophage.

In a yet further preferred embodiment, said second cysteine has beenartificially introduced into a modified variant of a wild type coatprotein of a bacteriophage.

Methods for achieving the artificial introduction according to thepresent invention are well-known to one of ordinary skill in the art,and involve standard cloning and/or mutagenesis techniques. Methods forthe construction of nucleic acid molecules encoding a modified variantof a wild type protein used in a method according to the presentinvention, for construction of vectors comprising said nucleic acidmolecules, for introduction of said vectors into appropriately chosenhost cells, for causing or achieving the expression of said fusionproteins are well-known in the art (see, e.g., Sambrook et al., 1989;Ausubel et al., 1999).

In another embodiment, the present invention relates to a method,wherein said second cysteine is present at, or in the vicinity of, theC-or the N-terminus of said member of the phage coat of saidbacteriophage particle.

The term “in the vicinity of” refers to a stretch of up to 15, or morepreferably, up to 10 amino acids, counted in both cases from either N-orC-terminus of said (poly)peptide/protein, provided that the N-orC-terminus is located at the outside of the bacteriophage.

Yet further preferred is a method, wherein said bacteriophage is afilamentous bacteriophage. Filamentous bacteriophage such as M13, fd, orf1 are well known to the artisan of ordinary skill in the art.

In the case of filamentous bacteriophage, a method is particularlypreferred, wherein said member of the protein coat of the bacteriophageparticle is or is derived from the wild type coat protein pIII.

Further preferred is a method, wherein said member of the protein coatof the bacteriophage particle is or is derived from the wild type coatprotein pIX. In the context of the present invention, the term “isderived” refers to a modification, wherein the modified protein iscapable of being incorporated into the protein coat of the bacteriophageparticle. Preferably, those parts of the modified protein correspondingto the wild type protein exhibit an amino acid identity exceeding about70%, preferably about 80%, most preferably about 90% compared to thecorresponding wild type sequence.

In a yet further preferred embodiment of the present invention, themethod comprises:

(a) providing a host cell harbouring a nucleic acid sequence comprisinga nucleic acid sequence encoding said (poly)peptide/protein;

(b) causing or allowing the expression of said nucleic acid sequence;and

(c) causing or allowing the production of bacteriophage particles insaid host cell.

In the context of the present invention, the term “causing or allowingthe expression” describes cultivating host cells under conditions suchthat nucleic acid sequence is expressed.

Methods for construction of nucleic acid molecules encoding a(poly)peptide/protein according to to the present invention, forconstruction of vectors comprising said nucleic acid molecules, forintroduction of said vectors into appropriately chosen host cells, forcausing or allowing the expression of (poly)peptides/proteins arewell-known in the art (see, e.g., Sambrook et al., 1989; Ausubel et al.,1999). Further well-known are methods for the introduction of geneticmaterial required for the generation of progeny bacteriophages orbacteriophage particles in appropriate host cells, and for causing orallowing the generation of said progeny bacteriophages or bacteriophageparticles (see, e.g., Kay et al., 1996). The step of causing or allowingthe production of bacteriophage particles may require the use ofappropriate helper phages, e.g. in the case of working with phagemids.

The steps (b) and (c) may be performed sequentially, in either order, orsimultaneously.

In a still further embodiment, said (poly)peptide/protein comprises animmunoglobulin or a functional fragment thereof.

In this context, “immunoglobulin” is used as a synonym for “antibody”.The term “functional fragment” refers to a fragment of an immunoglobulinwhich retains the antigen-binding moiety of an immunoglobulin.Functional immunoglobulin fragments according to the present inventionmay be Fv (Skerra & Plückthun, 1988), scFv (Bird et al., 1988; Huston etal., 1988), disulfide-linked Fv (Glockshuber et al., 1992; Brinkmann etal., 1993), Fab, F(ab′)₂ fragments or other fragments well-known to thepractitioner skilled in the art, which comprise the variable domain ofan immunoglobulin or immunoglobulin fragment.

Particularly preferred is an scFv or Fab fragment.

In a preferred embodiment, the present invention relates to a nucleicacid sequence encoding a modified variant of a wild type coat protein ofa bacteriophage, wherein said modified variant consists of:

(a) one or more parts of said wild type coat protein of a bacteriophage,wherein one of said parts comprises at least that part which causes orallows the incorporation of said coat protein into the phage coat; and

(b) between one and six additional amino acid residues not present atthe corresponding amino acid positions in a wild type coat protein of abacteriophage, wherein one of said additional amino acid residues is acysteine residue.

In the context of the present invention, a modified variant obtained bysubstitution of an amino acid residue in a wild type coat proteinsequence by a cysteine residue may be regarded as a variant composed oftwo parts of said wild type protein linked by an additional cysteineresidue. Correspondingly, variants of a wild type coat proteincomprising several mutations compared to the wild type sequence may beregarded as being composed of several wild type parts, wherein theindividual parts are linked by the mutated residues. However, saidvariant may also result from the addition of up to six residues,including a cysteine residue, to either C-and or N-terminus of the wildtype coat protein.

Further preferred is a nucleic acid sequence encoding a modified variantof a wild type coat protein of a bacteriophage, wherein said modifiedvariant consists of:

(a) one or more parts of said wild type coat protein of a bacteriophage,wherein one of said parts comprises at least that part which causes orallows the incorporation of said coat protein into the phage coat;

(b) between one and six additional amino acid residues not present atthe corresponding amino acid positions in a wild type coat protein of abacteriophage, wherein one of said additional amino acid residues is acysteine residue; and

(c) one or more peptide sequences for purification and/or detectionpurposes.

Particularly preferred are peptides comprising at least five histidineresidues (Hochuli et at., 1988), which are able to bind to metal ions,and can therefore be used for the purification of the protein to whichthey are fused (Lindner et al., 1992). Also provided for by theinvention are additional moieties such as the commonly used c-myc andFLAG tags (Hopp et al., 1988; Knappik & Plückthun, 1994), or theStrep-tag (Schmidt & Skerra, 1994; Schmidt et al., 1996).

The modified variant may further comprise amino acid residues requiredfor cloning, for expression, or protein transport. Amino acid residuesrequired for cloning may include residues encoded by nucleic acidsequences comprising recognition sequences for restriction endonucleaseswhich are incorporated in order to enable the cloning of the nucleicacid sequences into appropriate vectors. Amino acid residues requiredfor expression may include residues leading to increased solubility orstability of the (poly)peptide/protein. Amino acid residues required forprotein transport may include signalling sequences responsible for thetransport of the modified variant to the periplasm of E. coli, and/oramino acid residues facilitating the efficient cleavage of saidsignalling sequences. Further amino acid residues required for cloning,expression, protein transport, purification and/or detection purposesreferred to above are numerous moieties well known to the practitionerskilled in the art.

In another embodiment, the present invention relates to a vectorcomprising a nucleic acid sequence according to the present invention.

In a preferred embodiment, the vector further comprises one or morenucleic acid sequences encoding a (poly)peptide/protein comprising asecond cysteine residue.

In a most preferred embodiment, said (poly)peptide/protein comprises animmunoglobulin or a functional fragment thereof.

In the case of single-chain Fv antibody fragments referred tohereinabove, the vector comprises one nucleic acid sequence encoding theVH and VL domains linked by a (poly)peptide linker, and in the case ofFab antibody fragments, the vector comprises two nucleic acid sequencesencoding the VH-CH and the VL-CL chains.

In a further embodiment, the present invention relates to a host cellcontaining a nucleic acid sequence according to the present invention ora vector according to the present invention.

In the context of the present invention the term “host cell” may be anyof a number commonly used in the production of heterologous proteins,including but not limited to bacteria, such as Escherichia coli (Ge etal., 1995), or Bacillus subtilis (Wu et al., 1993), fungi, such asyeasts (Horwitz et al., 1988; Ridder et al., 1995) or filamentous fungus(Nyyssönen et al., 1993), plant cells (Hiatt & Ma, 1993; Whitelam etal., 1994), insect cells (Potter et al., 1993; Ward et al., 1995), ormammalian cells (Trill et al., 1995).

In a yet further preferred embodiment, the present invention relates toa modified variant of a wild type bacteriophage coat protein encoded bya nucleic acid sequence according to the present invention, a vectoraccording to the present invention or produced by a host cell accordingto the present invention.

In another embodiment, the present invention relates to a bacteriophageparticle displaying a (poly)peptide/protein on its surface obtainable bya method comprising:

causing or allowing the attachment of said (poly)peptide/protein afterexpression to a member of the protein coat of said bacteriophageparticle, wherein said attachment is caused by the formation of adisulfide bond between a first cysteine residue comprised in said(poly)peptide/protein and a second cysteine residue comprised in saidmember of the protein coat.

In another embodiment, the present invention relates to a bacteriophageparticle displaying a (poly)peptide/protein attached to its surface,wherein said attachment is caused by the formation of a disulfide bondbetween a first cysteine residue comprised in said (poly)peptide/proteinand a second cysteine residue comprised in a member of the protein coatof said bacteriophage particle.

In a preferred embodiment, the bacteriophage particle further contains avector comprising one or more nucleic acid sequences encoding said(poly)peptide/protein.

In a most preferred embodiment of the present invention, thebacteriophage particle contains a vector according to the presentinvention, wherein said vector comprises a nucleic acid sequenceencoding a modified wild type bacteriophage coat protein and furthermoreone or more nucleic acid sequences encoding a (poly)peptide/protein andmost preferably comprising at least a functional domain of animmunoglobulin.

The preferred embodiments of the method of the present inventionreferred to hereinabove mutatis mutandis apply to the bacteriophages ofthe present invention.

In a further embodiment, the present invention relates to a diversecollection of bacteriophage particles according to the presentinvention, wherein each of said bacteriophage particles displays a(poly)peptide/protein out of a diverse collection of(poly)peptides/proteins.

A “diverse collection of bacteriophage particles” may as well bereferred to as a “library” or a “plurality of bacteriophage particles”.Each member of such a library displays a distinct member of the library.

In the context of the present invention the term “diverse collection”refers to a collection of at least two particles or molecules whichdiffer in at least part of their compositions, properties, and/orsequences. For example, a diverse collection of (poly)peptides/proteinsis a set of (poly)peptides/proteins which differ in at least one aminoacid position of their sequence. Such a diverse collection of(poly)peptides/proteins can be obtained in a variety of ways, forexample by random mutagenesis of at least one codon of a nucleic acidsequence encoding a starting (poly)peptide/protein, by using error-pronePCR to amplify a nucleic acid sequence encoding a starting(poly)peptide/protein, or by using mutator strains as host cells in amethod according to the present invention. These and additional oralternative methods for the generation of diverse collections of(poly)peptides/proteins are well-known to one of ordinary skill in theart. A “diverse collection of bacteriophage particles” may be referredto as a library or a plurality of bacteriophage particles. Each memberof such a library displays a distinct member of the library.

In another embodiment, the invention relates to a method for obtaining a(poly)peptide/protein having a desired property comprising:

(a) providing the diverse collection of bacteriophage particlesaccording to the present invention; and

(b) screening said diverse collection and/or selecting from said diversecollection to obtain at least one bacteriophage particle displaying a(poly)peptide/protein having said desired property.

In the context of the present invention the term “desired property”refers to a predetermined property which one of the(poly)peptides/proteins out of the diverse collection of(poly)peptides/proteins should have and which forms the basis forscreening and/or selecting the diverse collection. Such propertiescomprise properties such as binding to a target, blocking of a target,activation of a target-mediated reaction, enzymatic activity, andfurther properties which are known to one of ordinary skill. Dependingon the type of desired property, one of ordinary skill will be able toidentify format and necessary steps for performing screening and/orselection.

Most preferred is a method, wherein said desired property is binding toa target of interest.

Said target of interest can be presented to said diverse collection ofbacteriophage particles in a variety of ways well known to one ofordinary skill, such as coated on surfaces for solid phase biopanning,linked to particles such as magnetic beads for biopanning in solution,or displayed on the surface of cells for whole cell biopanning orbiopanning on tissue sections. Bacteriophage particles having bound tosaid target can be recovered by a variety of methods well known to oneof ordinary skill, such as by elution with appropriate buffers, eitherby using a pH-or salt gradient, or by specific elution using solubletarget.

In a preferred embodiment, the method for obtaining a(poly)peptide/protein further comprises:

(ba) contacting said diverse collection of bacteriophage particles withthe target of interest;

(bb) eluting bacteriophage particles not binding to the target ofinterest;

(bc) eluting bacteriophage particles binding to the target of interestby treating the complexes of target of interest and bacteriophagesbinding to said target of interest formed in step (ba) under reducingconditions.

Under reducing conditions, such as by incubation with DTT, the disulfidebonds are cleaved, thus allowing to recover the specific bacteriophageparticles for further rounds of biopanning and/or for identification ofthe (poly)peptide/proteins specifically binding to said target.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a: Vector map of construct pMorphX7-hag2-LH.

FIG. 1b: Vector sequence of pMorphX7-hag2-LH (SEQ ID NO:35).

FIG. 2: Vector sequence of pTFT74-N1-hag-HIPM (SEQ ID NO:36)

FIG. 3: Vector sequence of pQE60-MacI (SEQ ID NO:37)

FIG. 4: Specific binding of scFv displayed on non-engineered phages.

Phages derived from constructs pMorphX7-MacI5-LCH, pMorphX7-MacI5-LHCand pMorphX7-MacI5-LH were produced by standard procedures andpre-incubated in PBSTM either with 5 mM DTT (+DTT) or without DTT. 5μg/well of specific antigen (MacI, dark columns) as well as unspecificcontrol antigen (BSA, light columns) were coated onto MaxisorpNunc-Immuno microtiter plates and incubated with 1×10¹⁰ phages/well,respectively. Bound phages were detected via anti-M13-HRP conjugate andBM blue soluble substrate. Phages derived from conventional phagedisplay vector pMorph13-MacI5 were used as control (3×10⁷ phages/well).Experimental details are given in Example 1.

FIG. 5: Specific binding of scFv displayed on non-engineered phages.

Phages derived from constructs pMorphX7-hag2-LCH, pMorphX7-hag2-LHC andpMorphX7-hag2-LH were produced by standard procedures and pre-incubatedin PBSTM either with 5 mM DTT (+DTT) or without DTT. 5 μg/well ofspecific antigen (N1-hag, dark columns) as well as unspecific controlantigen (BSA, light columns) were coated onto Maxisorp Nunc-Immunomicrotiter plates and incubated with 1×10¹⁰ phages/well, respectively.Bound phages were detected via anti-M13-FIRP conjugate and BM bluesoluble substrate. Phages derived from conventional phage display vectorpMorph13-hag2 were used as control (3×10⁷ phages/well). Experimentaldetails are given in Example 1.

FIG. 6a: Vector map of construct pBR-C-gIII.

FIG. 6b: Sequence of expression cassette for full length pIII with anN-terminal cysteine residue (C-gm) (SEQ ID NO:38).

FIG. 6c: Sequence of expression cassette for truncated pill with anN-terminal

cysteine residue (C-gIIICT) (SEQ ID NO:39).

FIG. 7a: Vector map of construct pMorph18-C-gIII-hag2-LHC.

FIG. 7b: Vector sequence of pMorph18-C-gIII-hag2-LHC (SEQ ID NO:40).

FIG. 8: Detection of scFv MacI-5 displayed on engineeredphages—Two-vector system.

Phages derived from constructs pMorphX7-MacI-5-LH/pBR-C-gIII (lanes 1 &5), pMorphX7-MacI-5-LHC/pBR-C-gIII (lanes 2 & 6), pMorphX7-MacI-5-LHC(lanes 3 & 7) and pMorphX7-MacI-5-LH (lanes 4 & 8) were produced bystandard procedures. 1‥5×10¹⁰ phages were pre-incubated in PBS with DTT(lanes 1-4) or without DTT (lanes 5-8). SDS loading buffer lackingreducing agents was added, phages were applied to an 4-15% SDS PAA Readygel and analysed in immunoblots. Detection of scFvs associated withphages was done via anti-FLAG Ml antibody, anti-mouse-IgG-AP conjugateand Fast BCTP/NPT substrate (6A) and via anti-pill antibody,anti-mouse-IgG-AP conjugate and Fast BCIP/NPT substrate (6B). Low rangemarker (Amersham #RPN756) is marked as M. Experimental details are givenin Example 2.1.

FIG. 9: Detection of scFvs Displayed on Engineered Phages—One-vectorsystem.

Phages derived from constructs pMorph18-C-gIII-hag2-LHC (lanes 1-8; 7A),pMorph18-C-gIII-AB1.1-LHC (lanes 1, 2, 5 and 6; 7B) andpMorph18-C-gIII-MacI-5-LHC (lanes 3, 4, 7 and 8; 7B) were produced bystandard procedures. 1-5×10¹⁰ phages were pre-incubated in PBS with DTT(lanes 1, 2, 5 and 6; 7A and lanes 1-4; 7B) or without DTT (lanes 3, 4,7 and 8; 7A and lanes 5-8; 7B). SDS loading buffer lacking reducingagents was added, phages were applied to an 4-15% SDS PAA Ready gel andanalysed in immunoblots. Detection of scFvs associated with phages wasdone via anti-FLAG M1 antibody, anti-mouse-IgG-AP conjugate and FastBCIP/NPT substrate (lanes 1-4; 7A) and via anti-pill antibody,anti-mouse-IgG-AP conjugate and Fast BCIP/NPT substrate (lanes 5-8; 7Aand lanes 1-8; 7B). Low range marker (Amersham #RPN756) is marked as M,Experimental details are given in Example 2.1.

FIG. 10: Specific binding of scFv displayed on engineeredphages—Comparison of the different two-vector systems

Phages derived from constructs pMorphX7-MacI-5-LHC/pBR-C-gIII (1),pMorphX7-MacI-5-LHC/pBR-C-gIIICT (2), pMorphX7-MacI-5-LHC/pUC-C-gIII(3), pMorphX7-MacI-5-LHC/pUC-C-gIIICT (4), pMorphX7-MacI-5-LHC (5),pMorphX7-MacI-5-LH (6) and the conventional phage display vectorpMorph13-MacI-5 (7) were produced by standard procedures. 5 μg ofspecific antigen (MacI) as well as unspecific control antigen (BSA, datanot shown) were coated onto Maxisorp Nunc-Immuno microtiter plates andincubated with a range of 6.4×10⁶ and 1×10¹¹ phages per well. Boundphages were detected via anti-M13-HRP conjugate and BM blue substrate.Experimental details are given in Example 2.1

FIG. 11: Specific binding of scFv displayed on engineeredphages—Comparison of the one- and two-vector system

Phages derived from constructs pMorphX7-MacI-5-LHC/pBR-C-gIII (1),pMorphX7-MacI-5-LHC/pBR-C-gIIICT (2), pMorph18-C-gIII-MacI-5-LHC (3),pMorph18-C-gIIICT-MacI-5-LHC (4) and pMorphX7-MacI-5-LHC (5) wereproduced by standard procedures. 5 μg of specific antigen (MacI, darkcolumns) as well as unspecific control antigen (BSA, light columns) werecoated onto Maxisorp Nunc-Immuno microtiter plates and incubated with1×10¹⁰ and 1×10⁹ phages, respectively. Bound phages were detected viaanti-M13-HRP conjugate and BM blue substrate. Experimental details aregiven in Example 2.1.

FIG. 12: Specific binding of scFv displayed on engineeredphages—Comparison of engineered gene III and gene IX proteins in theone-vector system

Phages derived from constructs pMorph18-C-gIII-MacI-5-LHC (1),pMorph18-C-gIIICT-MacI-5-LHC (2), pMorph18-C-gIX-MacI-5-LHC (3),pMorphX7-MacI-5-LHC (4) and the conventional phage display vectorpMorph13-MacI-5 (5) were produced by standard procedures. 5 μg ofspecific antigen (MacI, dark columns) as well as unspecific controlantigen (BSA, light columns) were coated onto Maxisorp Nunc-Immunomicrotiter plates and incubated with 1×10¹⁰, 1×10⁹ and 1×10⁸ phages,respectively. Bound phages were detected via anti-M13-HRP conjugate andBM blue substrate. Experimental details are given in Example 2.1.

FIG. 13: Specific binding of scFv displayed on engineered phages—Impactof DTT.

Phages derived from constructs pMorph18-C-gJII-MacJ-5-LHC (1),pMorph18-C-gIIICT-MacI-5-LHC (2), pMorph18-C-gIX-MacI-5-LHC (3),pMorphX7-MacI-5-LHC (4) and the conventional phage display vectorpMorph13-MacI-5 (5) were produced by standard procedures andpre-incubated in PBSTM either with 5 mM DTT (+) or without DTT (−). 5 μgof specific antigen (MacI, dark columns) as well as unspecific controlantigen (B SA, light columns) were coated onto Maxisorp Nunc-Immunomicrotiter plates and incubated with 1×10¹⁰ phages respectively. Boundphages were detected via anti-M13-HRP conjugate and BM blue substrate.Experimental details are given in Example 2.1.

FIG. 14: Specificity of selected scFvs—Panning of pre-selected poolsagainst N1-MacI.

scFvs selected after two rounds of cys-display panning against antigenN1-MacI from the κ-chain (1-5) and the λ-chain pool (6-8) were expressedaccording to standard procedures. 0.1 μg/well of milk powder (A), BSA(B), FITC-BSA (C, FJTC coupled to BSA), N1-hag (D), N1-Np50 (E) andN1-MacI (N1-MacI) was coated onto 384 well plates (Maxisorp; Nunc) andincubated with 10 μl scFv solution, respectively. Bound scFvs weredetected via a mixture of anti-Flag M1, anti-Flag M2 and anti-mouseIgG-AP conjugate as well as AttoPhos fluorescence substrate (Roche#1484281). Each scFv was tested in quadruplicates and mean values arepresented.

FIG. 15: Specificity of selected scFvs—Panning of pre-selected poolsagainst N1-Np50.

scFvs selected after two rounds of cys-display panning against antigenN1-Np50 (1-8) were expressed according to standard procedures. 0.1μg/well of milk powder (A), BSA (B), FITC-BSA (C, FITC coupled to BSA),N1-hag (D), N1-MacI (E) and N1-Np50 (N1-Np50) was coated onto 384 wellplates (Maxisorp; Nunc) and incubated with 10 μl scFv solution,respectively. Bound scFvs were detected via a mixture of anti-Flag M1,anti-Flag M2 and anti-mouse IgG-AP conjugate as well as AttoPhosfluorescence substrate (Roche #1484281). Each scFv was tested inquadruplicates and mean values are presented.

FIG. 16a: Vector map of construct pMorphX10-Fab-MacI5-VL-LHC-VH-FS.

FIG. 16b: Complete vector sequence of pMorphX10-Fab-MacI5-VL-LHC-VH-FS(SEQ NO:41).

FIG. 17: Detection of Fab ICAM1-C8 displayed on engineered phages

Phages derived from constructspMorphX10-Fab-ICAM1C8-VL-LHC-VH-MS/pBAD-SS-C-gIII (lanes 5, 6, 11, 12),pMorphX10-Fab-ICAM1C8-VL-LHC-VH-MS (lanes 3, 4, 9, 10) andpMorph18-Fab-ICAM1C8 (lanes 1, 2, 7, 8) were produced by standardprocedures. 1×10¹⁰ phages were pre-incubated in PBS with DTT (lanes 1-6)or without DTT (lanes 7-12). SDS loading buffer lacking reducing agentswas added, phages were applied to an 12% SDS PAA Ready gel and analysedin immunoblots. Detection was done via anti-pIII antibody,anti-mouse-IgG-HRP conjugate and BM Blue POD precipitating substrate.Low range molecular weight marker (Amersham Life Science #RPN756) ismarked as M. Experimental details are given in Example 2.2.

FIG. 18: Detection of Fab MacI-A8 displayed on engineered phages.

Phages derived from constructspMorphX10-Fab-MacIA8-VL-LHC-VH-FS/pBAD-SS-C-gIII (lanes 5, 6, 11, 12),pMorphX10-Fab-MacIA8-VL-LHC-VH-FS (lanes 3, 4, 9, 10) andpMorph18-Fab-MacIA8 (lanes 1, 2, 7, 8) were produced by standardprocedures. 1×10¹⁰ phages were pre-incubated in PBS with DTT (lanes 1-6)or without DTT (lanes 7-12). SDS loading buffer lacking reducing agentswas added, phages were applied to an 12% SDS PAA Ready gel and analysedin immunoblots. Detection was done via anti-pIII antibody,anti-mouse-IgG-HRP conjugate and BM Blue precipitating substrate. Lowrange molecular weight marker (Amersham Life Science #RPN756) is markedas M. Experimental details are given in Example 2.2.

FIG. 19: Specific binding of Fabs displayed on engineered phages—FabMacI-5.

Phages derived from constructspMorphX10-Fab-MacI5-VL-LHC-VH-FS/pBR-C-gIII (1),pMorphX10-Fab-MacI5-VL-C-VH-FS/pBR-C-gIII (2),pMorphX10-Fab-MacI5-VL-VH-CFS/pBR-C-gIII (3),pMorphX10-Fab-MacI5-VL-VH-LHC/pBR-C-gIII (4), pMorphX9-Fab-MacI5-FS (5),and the conventional phage display vector pMorph18-Fab-MacI5 (6) wereproduced by standard procedures. 5 μg/well of specific antigen (N1-MacI)were coated onto Maxisorp Nunc-Immuno microtiter plates and incubatedwith 1×10⁸ (light columns) and 1×10⁹ (dark columns) phages per well.Bound phages were detected via anti-M13-HRP conjugate and BM bluesoluble substrate. Each column represents the mean value of threeindependent phage preparations tested in duplicates. Experimentaldetails are given in Example 2.2.

FIG. 20: Specific binding of Fabs displayed on engineered phages—FabMacI-A8.

Phages derived from constructspMorphX10-Fab-MacIA8-VL-LHC-VH-FS/pBR-C-gIII (1),pMorphX10-Fab-MacIA8-VL-C-VH-FS/pBR-C-gIII (2),pMorphX10-Fab-MacIA8-VL-VH-CFS/pBR-C-gIII (3),pMorphX10-Fab-MacIA8-VL-VH-LHC/pBR-C-gIII (4), pMorphX9-Fab-MacIA8-FS(5), and the conventional phage display vector pMorph18-Fab-MacIA8 (6)were produced by standard procedures. 5 μg/well of specific antigen(N1-MacI) were coated onto Maxisorp Nunc-Immuno microtiter plates andincubated with 1×10⁹ (light columns) and 1×10¹⁰ (dark columns) phagesper well. Bound phages were detected via anti-M13-HRP conjugate and BMblue soluble substrate. Each column represents the mean value of threeindependent phage preparations tested in duplicates. Experimentaldetails are given in Example 2.2.

FIG. 21: Specific binding of Fabs displayed on engineered phages—FabICAM1-C8.

Phages derived from constructspMorphX10-Fab-ICAM1C8-VL-LHC-VH-MS/pBR-C-gIII (1),pMorphX10-Fab-ICAM1C8-VL-C-VH-MS/pBR-C-gIII (2),pMorphX10-Fab-ICAM1C8-VL-VH-CMS/pBR-C-gIII (3),pMorphX10-Fab-ICAM1C8-VL-VH-LHC/pBR-C-gIII (4), pMorphX9-Fab-ICAM1C8-MS(5), pMorphX9-Fab-ICAM1C8-MS/pBR-C-gIII (6) were produced by standardprocedures. 5 μg/well of specific antigen (ICAM1, dark columns) orunspecific antigen (BSA, light columns) were coated onto MaxisorpNunc-Immuno microtiter plates and incubated with 1×10⁹ phages per well.Bound phages were detected via anti-M13-HRP conjugate and BM bluesoluble substrate. Each column represents the mean value of one phagepreparation tested in duplicates. Experimental details are given inExample 2.2.

FIG. 22: Specific binding of Fabs displayed on engineered phages—Impactof DTT.

Phages derived from constructspMorphX10-Fab-MacI5-VL-LHC-VH-FS/pBR-C-gIII (1),pMorphX10-Fab-MacI5-VL-C-VH-FS/pBR-C-gIII (2),pMorphX10-Fab-MacI5-VL-VH-CFS/pBR-C-gIII (3),pMorphX10-Fab-MacI5-VL-VH-LHC/pBR-C-gIII (4), pMorphX9-Fab-MacI5-FS (5),and the conventional phage display vector pMorph18-Fab-MacI5 (6) wereproduced by standard procedures and pre-incubated in PBSTM either with10 MM DTT (+) or without DTT (−). 5 μg/well of specific antigen(N1-MacI, dark columns) as well as unspecific control antigen (BSA,light columns) were coated onto Maxisorp Nunc-Immuno microtiter platesand incubated with 1×10⁹ phages respectively. Bound phages were detectedvia anti-M13-HRP conjugate and BM blue substrate. Each column representsthe mean value of one phage preparation tested in duplicates.Experimental details are given in Example 2.2.

The examples illustrate the invention.

EXAMPLE 1 Display of (Poly)Peptides/Proteins on the Surface ofNon-Engineered Filamentous Bacteriophage Particles Via Formation ofDisulfide Bonds

In the following example, all molecular biology experiments areperformed according to standard protocols (Ausubel et al., 1999).

Construction of Vectors Expressing scFvs

All vectors used are derivatives of the high copy phagemid pMorphX7-LH(FIGS. 1a+b), a derivative of the pCAL vector series (WO 97/08320;Knappik et al., 2000). The expression cassette comprises the phoA signalsequence, a minimal binding site for the monoclonal antibody (mab)anti-FLAG M1 (Sigma #F-3040) (Knappik and Plückthun, 1994), a singlechain fragment (scFv), a short linker (PGGSG) and a 6× histidine tag(6His; Hochuli et al., 1988) (FIG. 1a). pMorphX7-LCH and pMorphX7-LHChave been generated by inserting oligonucleotide cassettes coding forCys-6His and 6His-Cys, respectively, between the unique AscI and HindIIIsites of pMorphX7-LH (FIG. 1a, Table 1). All vectors express solublescFv not genetically fused to any phage coat protein. The conventionalphage display vector pMorph13 which is based on the pCAL4 vectordescribed in WO 97/08320 and expresses a fusion of an scFv to theC-terminal part of phage protein pIII was used as positive control. ThescFvs have been exchanged between the respective vectors via the uniqueXbaI and EcoRI sites (c.f. FIG 1 a).

Description of the scfv—Antigen Interactions

All scFvs derive from a human combinatorial antibody library (HuCAL; WO97/08320; Knappik et al., 2000). The HuCAL VH and VL consensus genes(described in WO 97/08320), and the CDR3 sequences of the scFvs aregiven in Table 2. Clone hag2 was selected against a peptide frominfluenza virus hemagglutinine (aa 99-110 from hemagglutinine plusadditional flanking aa (shown in italics, CAGPYDVPDYASLRSHH (SEQ IDNO:14)), and clone MacI-5 against a fragment (MacI) of human CR-3 alphachain (SWISS-PROT entry P11215, aa 149-353 of human CR-3 alpha fused toa C-terminal sequence containing a 6× histidine tag). The correspondingantigens for ELISA and doped library experiments were obtained asfollows. The hag2 specific antigen N1-hag was produced using expressionvector pTFT74-N1-hag-HIPM, a derivative of vector pTFT74 (Freund et al.,1993) (FIG. 2). N1-hag comprises aa 1-82 of mature gene III protein ofphage M13 containing an additional methionine residue at the N-terminus(N1) fused to the amino acid sequence PYDVPDYASLRSHHHHHH (hag) (SEQ IDNO:1) comprising aa 99-110 from influenza virus hemagglutinine and a 6×histidine tag (in italics) Expression, purification and refolding ofN1-hag was done as described (Krebber, 1996, Krebber et al., 1997). Asantigen for MacI-5, a purified fragment (MacI) of human CR-3 alpha chain(SWISS-PROT entry P11215) fused to a C-terminal 6× histidine tag wasused. In detail, the expression cassette encodes an N-terminalmethionine, amino acids 149-353 of human CR-3 alpha and amino acidsIEGRHHHHIIH (SEQ ID NO:2). This cassette is flanked by uniquerestriction sites BspHI and HindIII and can e.g. be introduced into theunique NcoI and HindIII sites of pQE-60 (QIAGEN GmbH, Hilden, Germany),yielding expression vector pQE60-MacI (FIG. 3). Expression andpurification was performed using standard methods (The QIAexpressionist™3rd edition: A handbook for high-level expression and purification of6×His-tagged proteins (July 1998). QIAGEN GmbH, Hilden, Germany). Bovineserum albumin (BSA, Sigma #A7906) was used as negative control antigen.

Functionality of scFvs Displayed on Non-Engineered Phages

To demonstrate that the displayed scFvs are functional with respect torecognition of their specific antigens phage ELISAs were performed. Theanalysis was done for the two HuCAL scFvs hag2 and MacI-5. Threeexpression systems differing in the modules fused to the C-terminus ofthe scFv were analysed, namely pMorphX7-LH, pMorphX7-LHC andpMorphX7-LCH.

Phages were produced according to standard procedures using helper phageVCSM13 (Kay et al., 1996). Specific antigen or control antigen (BSA,Sigma #A7906) was coated for 12 h at 4° C. at a concentration of 5μg/well in PBS to Nunc Maxisorp microtiter plates (#442404).

Phages were pre-incubated in PBSTM (PBS containing 5% skimmed milkpowder and 0.1% Tween 20), either with or without 5 mM DTT, for 2 h atroom temperature before they were applied to the ELISA well coated withantigen at a concentration of 1×10¹⁰ phages per well except for pMorph13which was used at a concentration of 3×10⁷ phages per well. Afterbinding for 1 h at RT, non-specifically bound phages were washed awaywith PBS containing 0.05% Tween 20 and bound phages were detected inELISA using an anti-M13-HRP conjugate (Amersham Pharmacia Biotech#27-9421-01) and BM blue soluble (Boehringer Mannheim #1484281).Absorbance at 370 nm was measured. ELISA signals obtained with thespecific antigen were compared to those with the control antigen.Specific binding of scFv displaying phages to antigen could be shown. Asan example two of such ELISAs for scFvs hag2 and MacI-5 are presented inFIGS. 4 and 5, respectively. With phages derived from pMorphX7-LCH andpMorphX7-LHC signals between 1.9 and 5.8 times above background wereachieved. When 5 mM DTT was added to the phages prior to antigen bindingduring the pre-incubation step, the ELISA signal was decreased to almostbackground levels while DTT had no major effect on the conventionaldisplay phages (pMorph13).

Enrichment of Non-Engineered Phages Displaying scFv

To prove that non-engineered phages displaying scFvs can be enriched onspecific antigen a so called doped library experiment was performed.Specific phages were mixed with a high excess of unspecific phages andthree rounds of panning on specific antigen were performed. Theenrichment for specific phages was determined after each round. Theanalysis was done for the two HuCAL scFvs hag2 and MacI-5 in thepMorphX7-LHC vector.

pMorphX7-hag2-LHC and pMorphX7-MacI-5-LHC derived phages were mixed atratios of 1:10⁵ (pMorphX7-hag2-LHC panning) as well as 10⁵:1(pMorphX7-MacI-5-LHC panning). Three rounds of panning were performed onthe hag2 and MacI-5 specific antigen, respectively. Phages were preparedby standard procedure and pre-blocked by mixing 1:1 with PBSTM (PBS, 5%skimmed milk powder, 0.1% Tween20) and incubation for 2 h at RT. Wellsof a Nunc Maxisorp microtiter plate (#442404) were coated with specificantigen N1-hag (as well as BSA) at a concentration of 5 μg/well in PBSovernight at 4° C., and subsequently blocked with 400 μl PBSM (PBS, 5%skimmed milk powder) for 2 h at RT. For the first round, 10¹¹pre-blocked phages were applied per well and incubated for 1 h at RT ona microtiter plate shaker. Phage solution was removed and wells werewashed 3 times with PBST (PBS, 0.05% Tween20) and 3 times with PBS.Bound phages were eluted with 100 mM triethylamine according to standardprotocols and used for infection of TG1 cells. In addition, residualphages were eluted by direct infection of TG1 added to the wells. Aftereach round of panning on specific antigen the ratio of specific tounspecific phages was determined by analysing at least 46 independentinfected cells via PCR. The PCR was performed according to standardprotocols using single colonies as source of template andoligonucleotides specific for VH CDR3 and VL CDR3 of each scFv asprimers. After 3 rounds of panning, ˜4% positive clones (4 out of 93clones analysed) were obtained for the pMorphX7-hag2-LHC panning and˜90% positive clones (82 out of 91 clones analysed) were obtained forthe pMorphX7-MacI-5-LHC panning.

EXAMPLE 2 Display of (Poly)Peptides/Proteins on the Surface ofEngineered Filamentous Bacteriophage Particles Via Formation ofDisulfide Bonds EXAMPLE 2.1 Display of scFvs

Example 1 described above shows that functional scfvs can be displayedon non-engineered phages via disulfide bonds. This system can be furtherimproved, e.g. via engineering an exposed cysteine on a phage coatprotein. One candidate phage coat protein is protein III (pIII) which iscomposed of three domains N1, N2 and pIIICT. Possible sites forpositioning an unpaired cysteine residue are the linker regions betweenthe domains or the exposed N-terminus of the domain or the pIIICT in atruncated pIII version. A further example would be phage coat protein IX(pIX) where the cysteine could e.g. be linked to the N-terminus of thefull length protein. In principle the cassettes for expression of suchengineered proteins can be placed on the vector which is providing thescFv (one-vector system), or on a separate vector (two-vector system).

In the following we will describe experiments in which we engineeredboth a full length and a truncated pIII version as well as pIX) Theseproteins were co-expressed in the same bacterial cell together with thescfv, either from the same phagemid (pMorph18-C-gIII-scFv-LHCderivatives; one-vector system) or from a separate plasmid(pBR322-C-gIII or pUC 19-C-gill and derivatives; two-vector system).

Construction of Vectors Expressing scFvs and Engineered Phage CoatProteins

Phage coat protein expression cassettes for the two-vector system wereconstructed as follows: Two different expression cassettes flanked byunique NheI and HindIII restriction sites at the ends were madepositioning an unpaired cysteine residue at the exposed N-terminus ofthe N1-domain of frill length mature pIII (C-gIII) or at the N-terminusof the pIIICT domain of the truncated protein (amino acids 216 to 406 ofprotein pIII; C-gIIICT) (FIGS. 6b+c)). Both expression cassettes areunder the control of the lac promotor/operator region and comprise thesignal sequence ompA, amino acids DYCDIEF (SEQ ID NO:3) and the pill orpIIICT ORF (complete amino acid sequences are given in Table 3).Plasmids expressing the modified pIII proteins were obtained byinserting these NheI-HindIII cassettes into plasmid pBR322 and pUC19 viathe unique NheI and HindIII or XbaI and HindIII sites, respectively. Asan example, the vector map of pBR-C-gIII is depicted in FIG. 6a. Theresulting plasmids, pBR-C-gIII, pBR-C-gIIICT, pUC-C-gIII andpUC-C-gIIICT, were co-transformed with pMorphX7-LHC phagemids expressingthe modified scFv (Example 1) into E. coli TG1 selecting for bothantibiotic markers.

In the one-vector system both the modified phage coat proteins as wellas the modified scFv were expressed from a dicistronic phagemid undercontrol of the lac promotor/operator region. The first expressioncassette comprises the signal sequence ompA, amino acids DYCDIEF (SEQ IDNO:3) and the ORF for the respective phage coat protein or part thereofThe unpaired cysteine residue was linked to the exposed N-terminus ofthe N1-domain of full length mature pIII (C-gIII), to the N-terminus ofthe truncated protein III (amino acids 216 to 406 of protein pIII;C-gIIICT) and to the N-terminus of protein IX (C-gIX), respectively(amino acid sequences are given in Table 4). The second expressioncassette comprises the phoA signal sequence, the ORF of the respectivescFv, a short linker (PGGSG), a 6× histidine tag (6His; Hochuli et al.,1988) and the single cysteine residue (see pMorphX7-LHC, Table 1). Thecomplete vector sequence of pMorph18-C-gIII-hag2-LHC coding for modifiedfull length pIII as well as modified scFv hag2 and the respective vectormap are given in FIGS. 7a+b. The different phage coat proteins can beexchanged via EcoRI and StuI in a three fragment cloning procedure dueto a second EcoRI site at the 3′ end of the scFvs. The differentengineered scFvs can be cloned via the unique MfeI and HindIII sites. Aderivative of this vector, pMorph20-C-gIII-hag2-LHC, contains a uniqueEcoRI site at the 3′ end of the scFv while the second site (between theompA signal sequence and the gIII ORF) was deleted via silent PCRmutagenesis. This construct allows the cloning of scFvs or scFv poolsvia the unique SphI and EcoRI sites.

Attachment of scFvs to Phage Coat Proteins via Disulfide Bonds

Phage for biopanning applications can be produced using helper phageVCSM13 following standard protocols (Kay et al., 1996). In addition tohelper phage proteins, engineered phage coat protein and solublemodified scFv were co-expressed from the one-or two-vector systemsdescribed above. To demonstrate that the scFvs attach to the engineeredphage coat proteins via disulfide bridges and are incorporated intophage particles, scFv displaying phages were run on SDS PAGE undernon-reducing and reducing conditions. Western blot analysis wasperformed with anti-pIII and anti-Flag M1 antisera.

Phages were produced according to standard procedures using helper phageVCSM13 (Kay et al., 1996). Phages were pre-incubated in PBS with 5 mMDTT or without DTT (reducing and non-reducing conditions, respectively)for 30 minutes at room temperature before adding SDS loading bufferlacking reducing agents such as DTT or β-mercaptoethanol. 1-5×10¹⁰phages per lane were run on a 4-15% SDS PAGE (BioRad) and blotted ontoPVDF membranes. For the anti-pIII Western blot, the membrane was blockedin MPBST (PBS buffer containing 5% milk powder and 0.05% Tween20) anddeveloped with mouse anti-pIII (1:250 dilution; Mobitec) as primaryantibody, anti-mouse-IgG-AP conjugate (1:10000 dilution; SIGMA) assecondary antibody and BCIP/NPT tablets (SIGMA) as substrate. For theanti-Flag M1 Western blot, the membrane was blocked in MTBST-CaCl₂ (TBSbuffer containing 5% milk powder, 0.05% Tween20 and 1 mM CaCl₂) anddeveloped with mouse anti-Flag M1 (1:5000 dilution; Sigma) as primaryantibody, anti-mouse-IgG-AP conjugate (1:10000 dilution; SIGMA) assecondary antibody and BCIP/NPT tablets (SIGMA) as substrate.

Specific bands migrating at the height expected for the scFv linked tothe full length pIII could be shown both for the one-and two-vectorsystem. This signal can only be seen under non-reducing conditions anddisappears under DTT indicating that pIII and scFv are linked viadisulfide bonds (scFv-S—S-pIII). As an example for the two-vector systeman anti-Flag M1 and anti-pIII Western blot for scFv MacI-5 is shown inFIG. 8. When the scFv without additional cysteines (pMorph7x-MacI-5-LH)is expressed, only free scFv sticking to phages can be detected in theanti-Flag M1 Western blot (lane 8, FIG. 8A). When an additional cysteineis added to the scFv (pMorphX7-MacI-5-LHC), those bands can hardly beseen and a band migrating at the height of scFv dimers (scFv-S—S-scFvand/or (scFv-SH)₂) (and an unknown additional band (scFv-S-SX)) appear(lane 7, FIG. 8A). When the engineered scFvs are co-expressed with anengineered pIII containing an additional cysteine at the N-terminus(pMorphX7-MacI-5-LHC and pBR-C-gIII) the signals shift to a molecularweight corresponding to scFv-pIII heterodimers (scFv-S—S-pIII) (lane 6,FIG. 8A). As expected, this scFv-S—S-pIII signal cannot be seen whennon-engineered scFvs are co-expressed with the engineered pIII(pMorphX7-MacI-5-LH and pBR-C-gIII), although similar numbers of phageparticles are loaded in each lane (lane 5, FIG. 8A). In the presence ofreducing agents, the predominant signals are obtained from free scFvsfor all expression systems (lanes 1-4, FIG. 8A). In the anti-pIIIWestern blot, free protein III (pIII-SH and/or pIII) can be seen for allexpression systems both under reducing and non-reducing conditions(lanes 1-8, FIG. 8B). Specific bands migrating at the height expectedfor disulfide bonded protein III dimers (pIII-S—S-pIII) can only bedetected under non-reducing conditions when engineered protein III isexpressed (lanes 5 and 6 of FIG. 8B). Only when both engineered scFv andengineered protein III are co-expressed an additional band migrating atthe height of a disulfide-linked scFv and protein III (scFv-S—S-pIII)appears in addition to the disulfide bonded protein III dimers (lane 6,FIG. 8B). This band corresponds in size to the scFv-S—S-pIII signaldetected in the anti-Flag M1 Western (c.f. lane 6, FIG. 8A) and is DTTsensitive (c.f. lane 2, FIG. 8A). DTT sensitive bands migrating at theheight of disulfide-linked scFv and protein III and being detected bothwith anti-Flag M1 and anti-pIII antisera were also observed whenengineered scFv and engineered pIII were co-expressed from the samephagemid (pMorph18-C-pIII-scFv-LHC). As an example for this one-vectorsystem an anti-Flag M1 and anti-pIII Western blot for scFv hag2 andanti-pIII Western blots for scFvs AB 1.1 and MacI-5 are shown in FIGS.9A and 9B, respectively.

Functionality of scFvs Displayed on Engineered Phages

To show that the displayed scFvs are functional with respect torecognition of the specific antigen, phage ELISAs were performed. Theanalysis was done for the HuCAL scFvs MacI-5 and hag2. For thetwo-vector system, pMorphX7-LHC was co-transformed with pBR-C-gIII,pBR-C-gIIICT, pUC-C-gIII and pUC-C-gIIICT, respectively. Three differentone-vector constructs were analysed, namely pMorph18-C-gIII-scFv-LHC,pMorph18-C-gIIICT-scFv-LRC and pMorph18-C-gIX-scFv-LHC. To demonstratethat the scFvs attach to the engineered phage coat proteins viadisulfide bonds, phage ELISAs were performed both under non-reducing andreducing conditions.

Phages were produced according to standard procedures using helper phageVCSM13 and phage titers were determined (Kay et al., 1996). Specificantigen or control antigen (BSA, Sigma #A7906) was coated for 12 hoursat 4° C. at an amount of 5 μg/well in PBS to Nunc Maxisorp microtiterplates (# 442404) and blocked with PBS containing 5% skimmed milk powderfor 2 h. Phages were pre-incubated in PBS containing 2.5% skimmed milkpowder, 0.05% Tween 20, as well as 5 mM DTT, where applicable, for 2 hat room temperature before they were applied to the ELISA well coatedwith antigen at a concentration range between 6.4×10⁶ and 1×10¹¹ phagesper well. After binding for 1 h at RT, unspecifically bound phages werewashed away with PBS containing 0.05% Tween 20 and bound phages weredetected in ELISA using an anti-M13HRP conjugate (Amersham PharmaciaBiotech #27-9421-01) and BM blue soluble (Boehringer Mannheim #1484281).Absorbance at 370 nm was measured. ELISA signals obtained with thespecific antigen were compared to those with the control antigen.Specific binding of scFv displaying phages to antigen could be shown forthe C-gIII, C-gIIICT and C-gIX constructs in the one-vector format.C-gIII and C-gIIICT were also tested and shown to work in bothtwo-vector systems. As an example four such ELISAs for scFv MacI-5 arepresented in FIGS. 10-13. In all cases where phage coat proteins areengineered with an additional cysteine residue, ELISA signals aresignificantly increased compared to the pMorphX7-LHC signals where onlythe scFv carries an additional cysteine. When 5 mM DTT was added to thephages prior to antigen binding during the pre-incubation step, theELISA signal was decreased to almost background levels for all threeengineered phage coat constructs as well as the non-engineeredpMorphX7-LHC phages while DTT had no major effect on the conventionaldisplay phages (pMorph13; FIG. 13). This shows that for both thenon-engineered and engineered phages disulfide bonds are essential forthe functional display of scFvs on phages and thus for the specificbinding of scFv displaying phages to antigen.

Enrichment of Engineered Phages Displaying scfv in “Doped Library”Experiments

To prove that engineered phages displaying scFvs can be enriched onspecific antigen, a “doped library” experiment was performed: specificphages were mixed with a high excess of unspecific phages and threerounds of panning on specific antigen were performed The enrichment forspecific phages was determined after each round. The analysis was donefor the two HuCAL scFvs hag2 and MacI-5 in the pMorph18-C-gIII-scFv-LHCone-vector system.

pMorph18-C-gIII-hag2-LHC and pMorph18-C-gIII-MacI-5-LHC derived phageswere mixed at ratios of 1:10 (pMorph18-C-gIII-hag2-LHC panning) as wellas 10⁵:1 (pMorph18-C-gIII-MacI-5-LHC panning). Three rounds of panningwere performed on the hag2 and MacI-5 specific antigen, respectively.Phages were prepared by standard procedure and pre-blocked by mixing 1:1with PBSTM (PBS, 5% skimmed milk powder, 0.1% Tween20) and incubationfor 2 h at RT Wells of a Nunc Maxisorp plate (#442404) were coated withspecific antigen (as well as BSA) at a concentration of 5 μg/well in PBSovernight at 4° C., and subsequently blocked with 400 μl PBSM (PBS, 5%skimmed milk powder) for 2 h at RT. For the first round, 10¹⁰pre-blocked phages were applied per well and incubated for 1 h at RT ona microtiter plate shaker. Phage solution was removed and wells werewashed 3 times with PBST (PBS, 0.05% Tween20) and 3 times with PBS.Bound phages were eluted with 100 mM triethylamine according to standardprotocols and used for infection of TG1 cells. In addition, residualphages were eluted by direct infection of TG1 cells added to the wells.After each round of panning on specific antigen, the ratio of specificto unspecific phages was determined by analysing at least 91 independentinfected cells via PCR. The PCR was performed according to standardprotocols using single colonies as source of template andoligonucleotides specific for VH CDR3 and VL CDR3 of each scFv asprimers. After 2 rounds of panning, ˜0% positive clones (0 out of 93clones analysed) were obtained for the pMorph18-C-gIII-hag2-LHC panningand ˜3% positive clones (3 out of 91 clones analysed) were obtained forthe pMorph18-C-gIII-MacI-5-LHC panning. After 3 rounds of panning, thespecific clones were enriched to ˜79% (92 out of 117 clones analysed)for the pMorph18-C-gIII-hag2-LHC panning and to ˜100% (229 out of 229clones analysed) for the pMorph18-C-gIII-MacI-5-LHC panning.

Enrichment of Engineered Phages Displaying scFv in Pannings ofPre-Selected Pools

To prove that engineered phages displaying scFvs can be selected out ofa diverse pool, pannings of pre-selected libraries were performed. Poolsafter one round of conventional panning were subcloned into theengineered one-vector format and panning was continued for up to threefurther rounds (cys-display pannings).

Pannings were performed against the following antigens: (i) ICAM1comprising the extracellular part of mature ICAM1 (amino acids 1-454)plus amino acids CGRDYKDDDKHHHHHH (SEQ ID NO:4) containing the M2-Flagand the 6× histidine tag. (ii) N1-MacI comprising aa 1-82 of mature geneIII protein of phage M13 containing an additional methionine residue atthe N-terminus plus a short linker at the C-terminus (N1), fused to apolypeptide containing amino acids 149-353 of human CR-3 alpha chain(SWISS-PROT entry P11215) plus the C-terminal sequence IEGRHHHHHH (SEQID NO:2) which includes the 6× histidine tag; and (iii) N1-Np50comprising N1 fused to a polypeptide containing amino acids 2-366 ofhuman NFκB p50 plus amino acids EFSHHHHHH (SEQ ID NO:5) which includethe 6× histidine tag. Expression vectors for N1-MacI and N1-Np50 arebased on vector pTFT74 (Freund et al., 1993) (complete vector sequenceof pTFT74-N1-hag-HIPM given in FIG. 2). Expression, purification andrefolding was done as described (Krebber, 1996; Krebber et al., 1997).

Initially, one round of conventional panning of the antibody libraryHuCAL-scFv (WO 97/08320; Knappik et al., 2000) was performed accordingto standard protocols. Briefly, wells of Maxisorp microtiterplates(Nunc; #442404) were coated with the respective antigen dissolved in PBSand blocked with 5% skimmed milk powder in PBS. 1≧5×10¹² HuCAL-scFvphage were added for 1 h at 20° C. After several washing steps with PBST(PBS, 0.05% Tween20) and PBS, bound phage were eluted either with 100 mMtriethylamine or 100 mM glycine pH 2.2, immediately neutralised with 1 MTris/HCl pH 7.0 and used for infection of TG1 cells. In addition,residual phages were eluted by direct infection of TG1 cells added tothe wells. Pannings against N1-Np50 used the complete HuCAL-scFv library(κ and λ pools combined), in pannings against N1-MacI κ and λ lightchain pools were kept separated. Against ICAM1 one round of conventionalpanning of the λ Light chain part of HuCAL-scFv was performed andsubsequently the selected heavy chains again combined with the completelibrary of λ light chains. The resulting light chain optimised libraryhad a diversity of 1.4×10⁷.

The scFvs of the respective pools were subcloned into vectorpMorph20-C-gIII-scFv-LHC (one-vector format) via the unique SphI andEcoRI sites. Subsequently, three rounds of cys-display panning wereperformed. Phages were prepared by standard procedure and pre-blocked bymixing 1:1 with PBSTM (PBS, 5% skimmed milk powder, 0.1% Tween20) andincubated for 2 hrs at RT. Wells of a Nunc Maxisorp plate (#442404) werecoated with specific antigens at a concentration of 5 μg/well in PBSovernight at 4° C., and subsequently blocked with 400 μl PBSM (PBS, 5%skimmed milk powder) for 2 hrs at RT. For each round of cys-displaypanning, between 1×10¹⁰ and 4.5×10¹¹ pre-blocked phages were applied perwell and incubated for 1 h at RT on a microtiter plate shaker. Phagesolution was removed and wells were washed with PBST (PBS, 0.05%Tween20) and PBS with increasing stringency. The 1^(st) round was washed3× quick and 2×5 mm with PBST and PBS, respectively, the 2^(nd) round 1×quick and 4×5 mm with PBST and PBS, respectively, and the 3^(rd) round10× quick and 5×5 mm with PBST and PBS, respectively. Bound phages wereeluted with 100 mM triethylamine according to standard protocols andused for infection of TG1 cells. In addition, residual phages wereeluted by direct infection of TG1 cells added to the wells.

After each round of panning the number of antigen specific phages wasdetermined in an ELISA. N1-MacI, N1-Np50 and ICAM-Strep (comprisingamino acids 1-455 of mature ICAM1 plus SAWSHPQFEK (SEQ ID NO:6)containing the Strep-tag II) were used as antigens, respectively. Toensure high level expression the selected scFvs were subcloned intoexpression vector pMorphX7-FS (Table 1). Subcloning was done in twosteps. First the scFv fragments were isolated frompMorph20-C-gIII-scFv-LHC via AflII and EcoRI, then the fragments werere-digested with SphI and cloned into the EcoRI/SphI digestedpMorphX7-FS vector. This procedure ensured that only scFvs from vectorpMorph20-C-gIII-scFv-LHC were subcloned and excluded any contaminationwith scFvs from a conventional display or expression vector. Expressionof the scFvs and their testing in ELISA against the respective antigenswas done according to standard procedures. Clones which showed a signalof at least 3× above background in ELISA were considered positive. Theresults are summarised in Table 5. To prove that the selected scFvs bindstrongly and specifically to their respective antigen several positiveclones after 2 rounds of cys-display panning were selected and re-testedin quadruplicates in a specificity ELISA on six different antigens(FIGS. 14 & 15). Enrichment of antigen-specific binders could clearly bedemonstrated. Already after two rounds of cys-display panning of thepre-selected pools against N1-MacI, N1-Np50 and ICAM1 between 80% and97% of the tested clones were positive in ELISA. The affinity of some ofthe selected scFvs was determined in Biacore and Kd values in the rangeof 1 nM to 2.2 μM were determined. These results are similar to theenrichment factors and affinities obtained in a conventional panning ofthe respective pools performed in parallel. Some of the scFvs wereselected independently via cys-display as well as conventional panning.

Elution of Engineered Phages Displaying scFv via Reducing Agents

When screening phage display libraries in biopanning the problem remainshow to best recover phages which have bound to the desired target.Normally, this is achieved by elution with appropriate buffers, eitherby using a pH- or salt gradient, or by specific elution using solubletarget. However, the most interesting binders which bind with highaffinity to the target might be lost by that approach. One option withengineered cys-display phages is that the complexes of target andspecific bacteriophages can be treated with reducing agents, e.g. byincubation with DTT, to cleave the disulfide bond between scFv and phagecoat protein and to recover the specific bacteriophage particles.

Pannings of pre-selected pools against N1-MacI were performed accordingto the protocol described above. Phages were eluted either according tothe standard protocol with 100 mM triethylamine and a direct infectionof TG1 cells by residual phages, or by incubation of the wells with 20mM DTT in Tris buffer pH 8.0 for 10 min. After each round of panning thepool of selected scFvs was subcloned into expression vector pMorphX7-FSaccording to the two step procedure described above, and the number ofN1-MacI specific scFvs was determined in ELISA. To prove that theselected scFvs bind strongly and specifically to their respectiveantigen several positive clones were selected and re-tested intriplicates in a specificity ELISA. Enrichment of antigen-specificbinders could clearly be demonstrated for both elution procedures. Aftertwo rounds of panning of the MacI κ-pool and the MacI λ-pool a two-foldand five-fold, respectively, higher number of ELISA positive clones wasobtained for elution with reducing agents compared to conventionalelution.

EXAMPLE 2.2: Display of Fabs

Example 2.1 shows that functional single chain fragments can bedisplayed on engineered phages via disulfide bonds. In the following wewill describe experiments which show that the same is true for Fabs. Thecysteine was engineered at different positions of the Fab antibodyfragment. These Fabs were co-expressed in the same bacterial celltogether with engineered full length pIII based on a two-vector system.

Construction of Vectors Expressing Fabs and Engineered pIII

Heavy and light chains of the Fab fragment were expressed from adicistronic phagemid under control of the lac promotor/operator region.The first expression cassette comprises the signal sequence ompA and thevariable and constant domain of the light chain, the second expressioncassette comprises the signal sequence phoA and the variable andconstant domain of the heavy chain. Heavy and light chain are not linkedvia a disulfide bond. Modules containing the engineered cysteine werelocated at the C-terminus of either the light or the heavy chain.Several constructs differing in the amino acid composition of themodules were compared and are summarised in Table 6. As an example thecomplete vector sequence of pMorphX10-Fab-VL-LHC-VH-FS coding for themodified Fab MacI-5 and the respective vector map are given in FIGS.16a+b.

Two different plasmids were used for expression of full length pIII.Plasmid pBR-C-gIII was already described above. The respectiveexpression cassette comprises the signal sequence ompA, amino acidsDYCDIEF (SEQ ID NO:3) and the pill ORF under control of the lactosepromotor/operator region (Table 3, FIG. 6). Alternatively, plasmidpBAD-SS-C-gIII was used. Here the respective expression cassettecomprises the signal sequence of pIII, amino acids TMACDIEF (SEQ IDNO:7) and the pIII ORF under control of the arabinose promotor/operatorregion (Table 3). For construction of pBAD-SS-C-gIII the fragment codingfor the engineered cysteine plus pIII was amplified from pUG-C-gill viaPCR introducing the restriction sites NcoI and HindIII and cloned intothe commercially available vector pBAD/gIII A (Invitrogen). The plasmidspBR-C-gIII or pBAD-SS-C-gIII were co-transformed with the respectivepMorphX10-Fab phagemids expressing the modified Fab into E. coli TG1selecting for both antibiotic markers.

Description of the Fab-Antigen Interactions

Three different Fabs all deriving from a human combinatorial antibodylibrary (HuCAL; WO 97/08320; Knappik et al., 2000) were used forevaluation of Fab display on engineered phage. The HuCAL VH and VLconsensus genes (described in WO 97/08320), and the CDR3 sequences ofthe Fabs are given in Table 2. Fab MacI-5 is derived from the scFvMacI-5 described above and was converted into the Fab format (completevector map of pMorphX10-Fab-MacI5-VL-LHC-VH-FS is given in FIG. 16a).Fabs MacI-A8 and ICAM1-C8 were isolated directly from one of theHuCAL-Fab libraries. Clone MacI-A8 was selected against antigenMacI-Strep, which comprises an N-terminal methionine, amino acids149-353 of human CR-3 alpha chain (SWISS-PROT entry P11215) and aminoacids SAWSHPQFEK (SEQ ID NO:6) which include the Strep-tag II (Schmidtet al., 1996). Expression and purification were done according toSchmidt & Skerra (1994). N1-MacI was used as corresponding antigen forELISAs. N1-MacI is described above, and comprises an N-terminalmethionine, amino acids 1-82 of mature gene III protein of phage M13plus a short linker (N1), amino acids 149-353 of human CR-3 alpha chain(SWISS-PROT entry P11215) and amino acids IEGRHHHHHH (SEQ ID NO:2) whichinclude the 6× histidine tag. Clone ICAM1-C8 was selected againstantigen ICAM1 described above, which comprises the extracellular part ofmature ICAM1 (amino acids 1-454) plus amino acids CGRDYKDDDKHHHHHH (SEQID NO:4) containing the M2-Flag and the 6× histidine tags. The sameantigen was used for ELISA assays as well as in the doped libraryexperiment.

Attachment of Fabs to Phage Coat Proteins Via Disulfide Bonds

To demonstrate that the Fabs attach to pIII via disulfide bridges andare incorporated into phage particles, the respective phages were run onSDS PAGE under non-reducing and reducing conditions. Western blotanalyses were performed with antibodies detecting pill, the heavy chain,the lambda light and the kappa light chain, respectively. All constructsdescribed in table 6 were analysed and the results are shown forpMorphX10-ICAM1C8-VL-LHC-VH-FS plus pBAD-SS-C-gill andpMorphX10-MacIA8-VL-LHC-VH-MS plus pBAD-SS-C-gIII as an example in FIGS.17 and 18.

Phages were produced using helper phage VCSM13 following standardprotocols (Kay et al., 1996). In addition to helper phage proteins,engineered phage coat protein and soluble modified Fab were co-expressedfrom the two-vector system. Phages were pre-incubated in PBS with orwithout 20 mM DTT (reducing and non-reducing conditions, respectively)for 1 h at room temperature before adding SDS loading buffer lackingreducing agents such as DTT or β-mercaptoethanol. 1×10¹⁰ phages per lanewere run on a 12% SDS PAGE (BioRad) and blotted onto nitrocellulosemembranes (Schleicher & Schuell). For the anti-pIII Western blot, themembrane was blocked in MTBST (50 mM Tris buffer pH 7.4, containing 5%milk powder and 0.05% Tween20) and developed with mouse anti-pIII (1:250dilution, Mobitec) as primary antibody, anti-mouse-IgG-HRP conjugate(1:5000 dilution; SIGMA) as secondary antibody and BM Blue PODprecipitating (Roche #1442066) as substrate. For the detection of theheavy chain, kappa light and lambda light chain the primary antibodiesanti-Fd (1:5000 dilution; The binding site PC075), anti-human kappa(1:5000 dilution; Sigma K-4377) and anti-human lambda (1:500 dilution,Sigma L-6522) were used, respectively.

In the anti-pIII Western blots, free protein III (SH-pIII and/or pIII)can be detected for all expression systems both under reducing andnon-reducing conditions (FIGS. 17 and 18). When both engineered Fab andengineered protein III are co-expressed a signal migrating at the heightof a hetero-dimer of light chain and protein III (VL-CL-SS-pIII) appearsunder non-reducing conditions. In addition, a band migrating at theheight expected for disulfide bonded protein III dimers (pIII-SS-pIII)can be seen (lanes 11 & 12, FIGS. 17, 18). Both hetero-and homo-dimersdisappear when the samples are treated with DTT (lanes 5 & 6, FIGS. 17and 18) or when modified Fabs are coexpressed with non-engineered pIII(lanes 3, 4, 9 & 10, FIGS. 17 and 18). The hetero-dimer in this case oflight chain linked to the full length pIII could also be detected withanti-light chain antibodies in non-reducing gels but was absent underreducing conditions. In addition, a band migrating at the heightexpected for the homo-dimer of the light chain (VL-CL-SS-VL-CL) wasdetectable (data not shown). Similar results were obtained for allconstructs described in Table 6, and no significant difference betweenvectors pBR-C-gIII and pBAD-SS-C-gIII for supply of engineered pIII wasdetected (data not shown).

Functionality of Fabs Displayed on Engineered Phages

To show that the displayed Fabs are functional with respect torecognition of the specific antigen phage ELISAs were performed. Theanalysis was done for the HuCAL Fabs MacI-5, MacI-A8 and ICAM1-C8. Allformats differing in the position of cysteine at the Fab were compared(Table 6). To demonstrate that the Fabs attach to the engineered phagecoat proteins via disulfide bonds, phage ELISAs were performed bothunder non-reducing and reducing conditions.

The respective phagemids expressing the modified Fab were co-transformedwith pBR-C-gIII and phage production was performed under standardconditions (Kay et al., 1996). Conventional Fab display phages(pMorph18-Fab) served as positive control, a phagemid expression vectorfor expression of non-engineered Fab (pMorphX9-Fab-FS) served asnegative control. Specific antigen or control antigen (BSA, Sigma#A7906) was coated for 12 hours at 4° C. at an amount of 5 μg/well inPBS to Nunc Maxisorp microtiter plates (# 442404) and blocked with PBScontaining 5% skimmed milk powder, 0.05% Tween 20 for 1 h. Phages werepre-incubated in PBS containing 5% skimmed milk powder, 0.05% Tween 20,and 10 mM DTT where applicable for 1 h at room temperature before theywere applied to the ELISA well coated with antigen at a concentrationrange between 1×10⁸ and 1×10¹⁰ phages per well. After binding for 1 h atRT, unspecifically bound phages were washed away with PBS containing0.05% Tween 20 and PBS. Bound phages were detected in ELISA using ananti-M13-HRP conjugate (Amersham Pharmacia Biotech #27-9421-01) and BMblue soluble (Roche #1484281). Absorbance at 370 nm was measured. ELISAsignals obtained with the specific antigen were compared to those withthe control. Up to three independent phage preparations were analysedand mean values are given in FIGS. 19 to 22.

For all different two-vector formats specific binding of Fab displayingphages to antigen could be demonstrated (FIGS. 19-21, lanes 1-4). ForFab MacI-5 no significant difference between the four formats wasdetected (FIG. 19), while construct pMorphX10-Fab-VL-LHC-VH-FS showedreproducibly best results for Fab MacI-A8 and ICAM1-C8 (FIGS. 20 and21). When 10 mM DTT was added to the phages prior to antigen bindingduring the pre-incubation step, the ELISA signal was decreased to almostbackground levels for all cys-display phages while DTT had no majoreffect on conventional display phages (pMorph18-Fab) (shown for FabMacI-5 in FIG. 22). This shows that disulfide bonds are essential forthe functional display of Fabs on phages and thus for the specificbinding of Fab displaying phages to antigen.

Enrichment of Engineered Phages Displaying Fabs in Doped LibraryExperiments

To prove that engineered phages displaying Fabs can be enriched onspecific antigen, a “doped library” experiment was performed: specificphages were mixed with a high excess of unspecific phages and threerounds of panning on specific antigen were performed. The enrichment forspecific phages was determined after each round. The analysis was donefor the HuCAL Fab ICAM1-C8 in the two vector systempMorphX10-Fab-VL-LHC-VH-FS plus pBAD-SS-C-gIII.

Engineered phages displaying ICAM1-C8 and MacI-A8 were mixed at ratiosof 1:10⁵. Three rounds of panning were performed on the ICAM1 antigen.Phages were prepared by standard procedure, pre-blocked by mixing 1:1with PBSTM (PBS, 5% skimmed milk powder, 0.1% Tween 20) and incubatedfor 2 hrs at RT. Wells of a Nunc Maxisorp plate (#442404) were coatedwith specific antigen at a concentration of 5 μg/well in PBS overnightat 4° C., and subsequently blocked with 400 μl PBSM (PBS, 5% skimmedmilk powder) for 2 hrs at RT. For the first round, 10¹¹ pre-blockedphages were applied per well and incubated for 1 h at RT on a microtiterplate shaker. Phage solution was removed and wells were washed 3 timeswith PBST (PBS, 0.05% Tween 20; 1× quick, 2× 5 min) and 3 times with PBS(1× quick, 2×5 min). Bound phages were eluted with 100 mM triethylamineaccording to standard protocols. In addition, residual phages wereeluted by direct infection of cells added to the wells. As a directinfection of TG1 cells harbouring pBAD-SS-C-gIII was not efficientenough, eluted phages were used for infection of TG1 cells, amplifiedand than used for infection of TG1 cells harbouring pBAD-SS-C-gIII. Thusthe two-vector system was restored and the next round of panning wasperformed. While no difference between the two plasmids for expressionof engineered pIII (pBR-C-gIII and pBAD-SS-C-gIII) was observed withrespect to phage ELISA and WB, infection of TG1 cells harbouringpBR-C-gIII was not as efficient as infection of TG1 cells harbouringpBAD-SS-C-gIII. After each round of panning the ratio of specific tounspecific phages was determined by analysing at least 92 independentinfected cells via PCR. The PCR was performed according to standardprotocols using single colonies as source of template andoligonucleotides specific for the lambda light chain (priming inframework 4), the kappa light chain (priming in framework 3) and avector sequence upstream of the Fab fragment (commercial M13-revprimer,NEB) as primers. Fragments of roughly 420 bp length were expectedfor lambda Fabs (ICAM1-C8) and 290 bp for kappa Fabs (MacI-A8). After 2rounds of panning, 61% positive clones (57 out of 93 clones analysed)were obtained, which could be enriched to 100% (92 out of 92 clonesanalysed) after the third round.

REFERENCES

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TABLE 1 Amino acid sequence of ORF modules between the EcoRI and HindIIIsites of vectors pMorphX7-hag2-FS, pMorphX7-hag2-LH, pMorphX7-hag2-LCHand pMorphX7-hag2-LHC Construct EcoRI Module 1 AscI Module 2 HindIIIpMorphX7-FS EF DYKDDDDK GAP WSHPQFEK-stop stop (SEQ ID NO:8) (SEQ ID NO:pMorphX7-LH EF PGGSG GAP HHHHHH-stop stop (SEQ ID NO:10) (SEQ ID NO:11)pMorphX7-LCH EF PGGSG GAP CHHHHHH-stop stop (SEQ ID NO:10) (SEQ IDNO:12) pMorphX7-LHC EF PGGSG GAP HHHHHHC-stop stop (SEQ ID NO:10) (SEQID NO:13)

TABLE 2 Amino acid sequence of HuCAL scFvs and HuCAL Fabs* scFv antigenVH VH CDR3 VL VL CDR3 hag2 peptide of influenza virus VH3 RSGAYDY VK4QQYSSFPL hemagglutinine (SEQ ID NO: (SEQ ID NO: (CAGPYDVPDYASLRSHH) (SEQID NO: AB 1.1 12 amino acid peptide VH3 10 amino acid Vλ1 9 amino acidresidues residues MacI-5 fragment of human CR-3 VH2 FDPFFDSFFDY Vλ1QSYDQNALVE alpha chain (SEQ ID NO:17) (SEQ ID NO:18) MacI-A8 fragment ofhuman CR-3 VH3 HGYRKYYTDM Vκ1 HQVYSTSP alpha chain FDV (SEQ ID NO:20)(SEQ ID NO:19) ICAM1-C8 human ICAM1 VH2 FPYTYTIGFMID Vλ3 QSYDSGNL N (SEQID NO:22) (SEQ ID NO:21) *details are given in the Examples

TABLE 3 Amino acid sequence of engineered phage coat proteins of vectorpBR-C-gIII and derivatives EcoRV- Construct Signal Sequence EcoRIsequence HindIII pUC-C-gIII MKKTAIAIAVAL DYC DI EFAETVESCLAKPHTENSFTNYWKDD stop pBR-C-gIII AGFATVAQAKTLDRYANYEGCLWNATGVVVCT (ompA) GDETOCYGTWVPJGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPI PGYTYPNPLDGTYPPGTEQNPAMNPSLEESOPLNTFMFQThRfRNRQGA LTVYTGTVTOGTDPVKTYYOYTPVSSKAMYDAYWNGKFRDCAFHSGF NEDPFVCEYOGQSSDLPOPPVNAGGGSGGGSGGGSEGGGSEGGGSEGG GSEGGGSGGGSGSGDFDYEKMAN ANKGAMTENADENALOSDAKGKLDSVATDYGAAIDGFIGDVSGLANG NGATGDFAGSNSQMAQVGDGDNSPLMNNFROYLPSLPQSVECRPYVFG AGKPYEFSIDCDKTNLFRGVFAFLLYVATFMYVFSTFAMILRLNKES (SEQ ID NO:23) pUC-C- MKKTAIAIAVAL DYC DI EFNAGGGSGGGSGGGSEGGGSEGGGS stop gIIICT AGFATVAQA EGGGSEGGGSGGGSGSGDFDYEKpBR-C- (ompA) MANANKGAMTENADENALOSDAK gIIICT GKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSOMAOVGDG DNSPLMNNFRQYLPSLPOSVECRPFVFGAGKPYEFSTDCDKINLFRGVFA FLLYVATFMYVFSTFANILRNKES (SEQ ID NO:24)pBAD-SS- MIKKLLFAIPLVVTMA DY DI EF AETVESCLAKPHTENSFTNVWKDDKT stopC-gIII PFYSHS C LDRYANYEGCLWNATGVVVCTGDET (gIII) NcoIQCYGTWVPIGLAIPENEGGGSEGGGSE (StyI) GGGSEGGGTKPPEYGDTPIPGYTYINP /SphILDGTYPPGTEQNPANPNPSLEESQPLN TFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYDAYWNG KFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPVNAGGGSGGGSGGGSEGGGSEG GGSEGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTENADENALQSDAKGK LDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGDNSPLMN NFRQYLPSLPQSVECRYVFGAGKPYEFSIDCDKJNLFRGVFAFLLYVATFMYV FSTFANILRNKES (SEQ ID NO:25) The engineeredCys is written in bold Sequence of wild type phage coat proteins isunderlined

TABLE 4 Amino acid sequence of engineered phage coat proteins of vectorpMorph18- C-gIII-scFv-LHC and derivatives OmpA EcoRV- Construct SignalSequence EcoRI sequence StuI pMorph18-C- MKKTAIAIAVAL DY DI EFAETVESCLAKPHTENSFTNVSKD stop gIII-scFv- AGFATVAQA CDKTLDRYANYEGCLWNATGVVV LHC CTGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEY GDTPIPGYTYIMNPLDGTYPPGTEQNPANPNPSLEESOPLNTFMQNNR FRNRQGALTVYTGTVTOGTDPVK TYYOYTPVSSKAMYDAYWNGKFRDCAFHSGFNEDPFVCEYOGQSSD LPQPPVNAGGGSGGGSGGGSEEG GSEGGGSEGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTENADE NALOSDAKGKLDSVATDYGAAID GFIGDVSGLANGNGATGDFAGSNSQMAOVGDGDNSPLMINMBRQYL PSLPQSVECRPYVFGAGKYYEFSIDCDKTNLFRGVFAFLLYVATFMYXTF STFANILRNKES (SEQ lID NO:26) pMorph18-C-MKKTAIAIAVAL DY DI EF NAGGGSGGGSGSEGGGSEGGG stop gIIICT-scFv- AGFATVAQAC SEGGGSEGGGSGGGSGSGDFDYE LHC KMANANKGAMTENADENAIOSD AKGKLDSVATDYGAAIDGFIGDVS GLANGNGATGDFAGSNSQMAOV GDGDNSPLMNNFRQYLPSLPQSVECRPFVFGAGKPYEFSIDCDKINLF RGVFAFLLYVATFMYVFSTFANIL RINKES (SEQ ID NO:27)pMorph18-C- MIKKTAIAIAVAL DY DI EF GGGGSMSVLVYSFASFVLGWCLR stopgIX-scFv- AGFATVAQA C SGITYFTRLMETSS LHC (SEQ ID NO:28) The engineeredCys is written in bold Sequence of wild type phage coat proteins isunderlined

TABLE 5 Cys-display panning of pre-selected pools Preselected # of # ofPanning Pool clones^(a) positives^(b) Format round 1 round 2 round 3N1-MacI   2 × 10⁵ 3/186 = Cys-display 78/279 = 28% 89/93 = 96% 92/93 =99% κ chains 2% conventional 10/93 = 11% 71/93 = 76% nd N1-MacI   4 ×10⁴ 4/186 = Cys-display 72/279 = 26% 90/93 = 97% 90/93 = 97% λ chains 2%conventional 34/93 = 37% 87/93 = 94% nd N1-Np50   5 × 10⁴ 0/186 =Cys-display 17/93 = 18% 244/279 = 87% nd 0% conventional 51/93 = 55%86/93 = 92% nd ICAM1 1.4 × 10⁷ nd Cys-display 4/186 = 2% 149/186 = 80%nd nd: not determined ^(a)N1-MacI, N1-Np50: number of clones after oneround of conventional panning; ICAM1: diversity of the light chainoptimised pool. ^(b)number of ELISA positives of the respectivepre-selected pools.

TABLE 6 Amino acid sequence of modules of engineered Fab fragment Moduleat the light chain Module at the heavy chain Construct elements aminoacids elements amino acids pMorphX10-Fab- linker- SPGGSG-GAP- linker EF-VL-LHC-VH-FS histidine tag- HHHHHH- Flag tag-linker DYKDDDDK-GAP-cysteine C-stop Strep-tag II WSLIPQFEK-stop (SEQ ID NO:29) (SEQ IDNO:30) pMorphX10-Fab- linker- SPGGSG-GAP- linker- EF- VL-LHC-VH-MShistidine tag- HHHHHH- myc tag-linker- EQKLISEEDLN-GAP- cysteine C-stopStrep-tag II WSHIPQFEK-stop (SEQ ID NO:29) (SEQ ID NO:31) pMorphX10-Fab-cysteine deletion of A- linker- EF- VL-C-VH-FS C-stop (κ-chains) Flagtag-linker- DYKDDDDK-GAP- CS-stop (λ-chains) Strep-tag II WSIIPQFEK-stop(SEQ ID NO:30) pMorphX10-Fab- cysteine deletion of A- linker- EF-VL-C-VH-MS C-stop (κ-chains) myc tag-linker- EQKLISEEDLN-GAP- CS-stop(λ-chains) Strep-tag II WSFEPQFEK-stop (SEQ ID NO:3 1) pMorphX10-Fab- —— linker- EF-PGGSG-GAP- VL-VH-LHC histidine tag- HHHHHH- cysteine C-stop(SEQ ID NO:32) pMorphX10-Fab- — — cysteine-linker- C-EF- VL-VH-CFS Flagtag-linker- DYKDDDDK-GAP- Strep-tag II WSHPQFEK-stop (SEQ ID NO:33)pMorphX10-Fab- — — cysteine-linker- C-EF- VL-VH-CMS myc tag-linker-EQKLISEEDLN-GAP- Strep-tag II WSHPQFEK-stop (SEQ ID NO:34) Theengineered cysteine is written in bold

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 41 <210> SEQ ID NO 1 <211> LENGTH: 18<212> TYPE: PRT <213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 1Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Ar #g Ser His His His His1               5    #                10   #                15 His His<210> SEQ ID NO 2 <211> LENGTH: 10 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 2 Ile Glu Gly Arg His His His His His His1               5    #                10 <210> SEQ ID NO 3<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: synthetic       module <400> SEQUENCE: 3Asp Tyr Cys Asp Ile Glu Phe 1               5 <210> SEQ ID NO 4<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: synthetic       module <400> SEQUENCE: 4Cys Gly Arg Asp Tyr Lys Asp Asp Asp Lys Hi #s His His His His His1               5    #                10   #                15<210> SEQ ID NO 5 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 5 Glu Phe Ser His His His His His His1               5 <210> SEQ ID NO 6 <211> LENGTH: 10 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 6 Ser Ala Trp Ser His Pro Gln Phe Glu Lys1               5    #                10 <210> SEQ ID NO 7<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: synthetic       module <400> SEQUENCE: 7Thr Met Ala Cys Asp Ile Glu Phe 1               5 <210> SEQ ID NO 8<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: synthetic       module <400> SEQUENCE: 8Asp Tyr Lys Asp Asp Asp Asp Lys 1               5 <210> SEQ ID NO 9<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: synthetic       module <400> SEQUENCE: 9Trp Ser His Pro Gln Phe Glu Lys 1               5 <210> SEQ ID NO 10<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: synthetic       module <400> SEQUENCE: 10 Pro Gly Gly Ser Gly1               5 <210> SEQ ID NO 11 <211> LENGTH: 6 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 11 His His His His His His1               5 <210> SEQ ID NO 12 <211> LENGTH: 7 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 12 Cys His His His His His His1               5 <210> SEQ ID NO 13 <211> LENGTH: 7 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 13 His His His His His His Cys1               5 <210> SEQ ID NO 14 <211> LENGTH: 17 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 14Cys Ala Gly Pro Tyr Asp Val Pro Asp Tyr Al #a Ser Leu Arg Ser His1               5    #                10   #                15 His<210> SEQ ID NO 15 <211> LENGTH: 7 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 15 Arg Ser Gly Ala Tyr Asp Tyr1               5 <210> SEQ ID NO 16 <211> LENGTH: 8 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 16 Gln Gln Tyr Ser Ser Phe Pro Leu1               5 <210> SEQ ID NO 17 <211> LENGTH: 11 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 17Phe Asp Pro Phe Phe Asp Ser Phe Phe Asp Ty #r 1               5   #                10 <210> SEQ ID NO 18 <211> LENGTH: 10 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 18 Gln Ser Tyr Asp Gln Asn Ala Leu Val Glu1               5    #                10 <210> SEQ ID NO 19<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: synthetic       module <400> SEQUENCE: 19His Gly Tyr Arg Lys Tyr Tyr Thr Asp Met Ph #e Asp Val1               5    #                10 <210> SEQ ID NO 20<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: synthetic       module <400> SEQUENCE: 20His Gln Val Tyr Ser Thr Ser Pro 1               5 <210> SEQ ID NO 21<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: synthetic       module <400> SEQUENCE: 21Phe Pro Tyr Thr Tyr His Gly Phe Met Asp As #n 1               5   #                10 <210> SEQ ID NO 22 <211> LENGTH: 8 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 22 Gln Ser Tyr Asp Ser Gly Asn Leu1               5 <210> SEQ ID NO 23 <211> LENGTH: 434 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 23Met Lys Lys Thr Ala Ile Ala Ile Ala Val Al #a Leu Ala Gly Phe Ala1               5    #                10   #                15Thr Val Ala Gln Ala Asp Tyr Cys Asp Ile Gl #u Phe Ala Glu Thr Val            20       #            25       #            30Glu Ser Cys Leu Ala Lys Pro His Thr Glu As #n Ser Phe Thr Asn Val        35           #        40           #        45Trp Lys Asp Asp Lys Thr Leu Asp Arg Tyr Al #a Asn Tyr Glu Gly Cys    50               #    55               #    60Leu Trp Asn Ala Thr Gly Val Val Val Cys Th #r Gly Asp Glu Thr Gln65                   #70                   #75                   #80Cys Tyr Gly Thr Trp Val Pro Ile Gly Leu Al #a Ile Pro Glu Asn Glu                85   #                90   #                95Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gl #y Gly Gly Ser Glu Gly            100       #           105       #           110Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Th #r Pro Ile Pro Gly Tyr        115           #       120           #       125Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pr #o Pro Gly Thr Glu Gln    130               #   135               #   140Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Gl #u Ser Gln Pro Leu Asn145                 1 #50                 1 #55                 1 #60Thr Phe Met Phe Gln Asn Asn Arg Phe Arg As #n Arg Gln Gly Ala Leu                165   #               170   #               175Thr Val Tyr Thr Gly Thr Val Thr Gln Gly Th #r Asp Pro Val Lys Thr            180       #           185       #           190Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Lys Al #a Met Tyr Asp Ala Tyr        195           #       200           #       205Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe Hi #s Ser Gly Phe Asn Glu    210               #   215               #   220Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Se #r Ser Asp Leu Pro Gln225                 2 #30                 2 #35                 2 #40Pro Pro Val Asn Ala Gly Gly Gly Ser Gly Gl #y Gly Ser Gly Gly Gly                245   #               250   #               255Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Se #r Glu Gly Gly Gly Ser            260       #           265       #           270Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Se #r Gly Asp Phe Asp Tyr        275           #       280           #       285Glu Lys Met Ala Asn Ala Asn Lys Gly Ala Me #t Thr Glu Asn Ala Asp    290               #   295               #   300Glu Asn Ala Leu Gln Ser Asp Ala Lys Gly Ly #s Leu Asp Ser Val Ala305                 3 #10                 3 #15                 3 #20Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe Il #e Gly Asp Val Ser Gly                325   #               330   #               335Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp Ph #e Ala Gly Ser Asn Ser            340       #           345       #           350Gln Met Ala Gln Val Gly Asp Gly Asp Asn Se #r Pro Leu Met Asn Asn        355           #       360           #       365Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gln Se #r Val Glu Cys Arg Pro    370               #   375               #   380Tyr Val Phe Gly Ala Gly Lys Pro Tyr Glu Ph #e Ser Ile Asp Cys Asp385                 3 #90                 3 #95                 4 #00Lys Ile Asn Leu Phe Arg Gly Val Phe Ala Ph #e Leu Leu Tyr Val Ala                405   #               410   #               415Thr Phe Met Tyr Val Phe Ser Thr Phe Ala As #n Ile Leu Arg Asn Lys            420       #           425       #           430 Glu Ser<210> SEQ ID NO 24 <211> LENGTH: 219 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 24Met Lys Lys Thr Ala Ile Ala Ile Ala Val Al #a Leu Ala Gly Phe Ala1               5    #                10   #                15Thr Val Ala Gln Ala Asp Tyr Cys Asp Ile Gl #u Phe Asn Ala Gly Gly            20       #            25       #            30Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gl #u Gly Gly Gly Ser Glu        35           #        40           #        45Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gl #y Gly Gly Ser Gly Gly    50               #    55               #    60Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu Ly #s Met Ala Asn Ala Asn65                   #70                   #75                   #80Lys Gly Ala Met Thr Glu Asn Ala Asp Glu As #n Ala Leu Gln Ser Asp                85   #                90   #                95Ala Lys Gly Lys Leu Asp Ser Val Ala Thr As #p Tyr Gly Ala Ala Ile            100       #           105       #           110Asp Gly Phe Ile Gly Asp Val Ser Gly Leu Al #a Asn Gly Asn Gly Ala        115           #       120           #       125Thr Gly Asp Phe Ala Gly Ser Asn Ser Gln Me #t Ala Gln Val Gly Asp    130               #   135               #   140Gly Asp Asn Ser Pro Leu Met Asn Asn Phe Ar #g Gln Tyr Leu Pro Ser145                 1 #50                 1 #55                 1 #60Leu Pro Gln Ser Val Glu Cys Arg Pro Phe Va #l Phe Gly Ala Gly Lys                165   #               170   #               175Pro Tyr Glu Phe Ser Ile Asp Cys Asp Lys Il #e Asn Leu Phe Arg Gly            180       #           185       #           190Val Phe Ala Phe Leu Leu Tyr Val Ala Thr Ph #e Met Tyr Val Phe Ser        195           #       200           #       205Thr Phe Ala Asn Ile Leu Arg Asn Lys Glu Se #r     210              #   215 <210> SEQ ID NO 25 <211> LENGTH: 432 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 25Met Lys Lys Leu Leu Phe Ala Ile Pro Leu Va #l Val Pro Phe Tyr Ser1               5    #                10   #                15His Ser Thr Met Ala Cys Asp Ile Glu Phe Al #a Glu Thr Val Glu Ser            20       #            25       #            30Cys Leu Ala Lys Pro His Thr Glu Asn Ser Ph #e Thr Asn Val Trp Lys        35           #        40           #        45Asp Asp Lys Thr Leu Asp Arg Tyr Ala Asn Ty #r Glu Gly Cys Leu Trp    50               #    55               #    60Asn Ala Thr Gly Val Val Val Cys Thr Gly As #p Glu Thr Gln Cys Tyr65                   #70                   #75                   #80Gly Thr Trp Val Pro Ile Gly Leu Ala Ile Pr #o Glu Asn Glu Gly Gly                85   #                90   #                95Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gl #y Ser Glu Gly Gly Gly            100       #           105       #           110Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Il #e Pro Gly Tyr Thr Tyr        115           #       120           #       125Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gl #y Thr Glu Gln Asn Pro    130               #   135               #   140Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gl #n Pro Leu Asn Thr Phe145                 1 #50                 1 #55                 1 #60Met Phe Gln Asn Asn Arg Phe Arg Asn Arg Gl #n Gly Ala Leu Thr Val                165   #               170   #               175Tyr Thr Gly Thr Val Thr Gln Gly Thr Asp Pr #o Val Lys Thr Tyr Tyr            180       #           185       #           190Gln Tyr Thr Pro Val Ser Ser Lys Ala Met Ty #r Asp Ala Tyr Trp Asn        195           #       200           #       205Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gl #y Phe Asn Glu Asp Pro    210               #   215               #   220Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser As #p Leu Pro Gln Pro Pro225                 2 #30                 2 #35                 2 #40Val Asn Ala Gly Gly Gly Ser Gly Gly Gly Se #r Gly Gly Gly Ser Glu                245   #               250   #               255Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gl #y Gly Gly Ser Glu Gly            260       #           265       #           270Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly As #p Phe Asp Tyr Glu Lys        275           #       280           #       285Met Ala Asn Ala Asn Lys Gly Ala Met Thr Gl #u Asn Ala Asp Glu Asn    290               #   295               #   300Ala Leu Gln Ser Asp Ala Lys Gly Lys Leu As #p Ser Val Ala Thr Asp305                 3 #10                 3 #15                 3 #20Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly As #p Val Ser Gly Leu Ala                325   #               330   #               335Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala Gl #y Ser Asn Ser Gln Met            340       #           345       #           350Ala Gln Val Gly Asp Gly Asp Asn Ser Pro Le #u Met Asn Asn Phe Arg        355           #       360           #       365Gln Tyr Leu Pro Ser Leu Pro Gln Ser Val Gl #u Cys Arg Pro Tyr Val    370               #   375               #   380Phe Gly Ala Gly Lys Pro Tyr Glu Phe Ser Il #e Asp Cys Asp Lys Ile385                 3 #90                 3 #95                 4 #00Asn Leu Phe Arg Gly Val Phe Ala Phe Leu Le #u Tyr Val Ala Thr Phe                405   #               410   #               415Met Tyr Val Phe Ser Thr Phe Ala Asn Ile Le #u Arg Asn Lys Glu Ser            420       #           425       #           430<210> SEQ ID NO 26 <211> LENGTH: 434 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 26Met Lys Lys Thr Ala Ile Ala Ile Ala Val Al #a Leu Ala Gly Phe Ala1               5    #                10   #                15Thr Val Ala Gln Ala Asp Tyr Cys Asp Ile Gl #u Phe Ala Glu Thr Val            20       #            25       #            30Glu Ser Cys Leu Ala Lys Pro His Thr Glu As #n Ser Phe Thr Asn Val        35           #        40           #        45Trp Lys Asp Asp Lys Thr Leu Asp Arg Tyr Al #a Asn Tyr Glu Gly Cys    50               #    55               #    60Leu Trp Asn Ala Thr Gly Val Val Val Cys Th #r Gly Asp Glu Thr Gln65                   #70                   #75                   #80Cys Tyr Gly Thr Trp Val Pro Ile Gly Leu Al #a Ile Pro Glu Asn Glu                85   #                90   #                95Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gl #y Gly Gly Ser Glu Gly            100       #           105       #           110Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Th #r Pro Ile Pro Gly Tyr        115           #       120           #       125Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pr #o Pro Gly Thr Glu Gln    130               #   135               #   140Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Gl #u Ser Gln Pro Leu Asn145                 1 #50                 1 #55                 1 #60Thr Phe Met Phe Gln Asn Asn Arg Phe Arg As #n Arg Gln Gly Ala Leu                165   #               170   #               175Thr Val Tyr Thr Gly Thr Val Thr Gln Gly Th #r Asp Pro Val Lys Thr            180       #           185       #           190Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Lys Al #a Met Tyr Asp Ala Tyr        195           #       200           #       205Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe Hi #s Ser Gly Phe Asn Glu    210               #   215               #   220Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Se #r Ser Asp Leu Pro Gln225                 2 #30                 2 #35                 2 #40Pro Pro Val Asn Ala Gly Gly Gly Ser Gly Gl #y Gly Ser Gly Gly Gly                245   #               250   #               255Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Se #r Glu Gly Gly Gly Ser            260       #           265       #           270Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Se #r Gly Asp Phe Asp Tyr        275           #       280           #       285Glu Lys Met Ala Asn Ala Asn Lys Gly Ala Me #t Thr Glu Asn Ala Asp    290               #   295               #   300Glu Asn Ala Leu Gln Ser Asp Ala Lys Gly Ly #s Leu Asp Ser Val Ala305                 3 #10                 3 #15                 3 #20Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe Il #e Gly Asp Val Ser Gly                325   #               330   #               335Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp Ph #e Ala Gly Ser Asn Ser            340       #           345       #           350Gln Met Ala Gln Val Gly Asp Gly Asp Asn Se #r Pro Leu Met Asn Asn        355           #       360           #       365Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gln Se #r Val Glu Cys Arg Pro    370               #   375               #   380Tyr Val Phe Gly Ala Gly Lys Pro Tyr Glu Ph #e Ser Ile Asp Cys Asp385                 3 #90                 3 #95                 4 #00Lys Ile Asn Leu Phe Arg Gly Val Phe Ala Ph #e Leu Leu Tyr Val Ala                405   #               410   #               415Thr Phe Met Tyr Val Phe Ser Thr Phe Ala As #n Ile Leu Arg Asn Lys            420       #           425       #           430 Glu Ser<210> SEQ ID NO 27 <211> LENGTH: 219 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 27Met Lys Lys Thr Ala Ile Ala Ile Ala Val Al #a Leu Ala Gly Phe Ala1               5    #                10   #                15Thr Val Ala Gln Ala Asp Tyr Cys Asp Ile Gl #u Phe Asn Ala Gly Gly            20       #            25       #            30Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gl #u Gly Gly Gly Ser Glu        35           #        40           #        45Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gl #y Gly Gly Ser Gly Gly    50               #    55               #    60Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu Ly #s Met Ala Asn Ala Asn65                   #70                   #75                   #80Lys Gly Ala Met Thr Glu Asn Ala Asp Glu As #n Ala Leu Gln Ser Asp                85   #                90   #                95Ala Lys Gly Lys Leu Asp Ser Val Ala Thr As #p Tyr Gly Ala Ala Ile            100       #           105       #           110Asp Gly Phe Ile Gly Asp Val Ser Gly Leu Al #a Asn Gly Asn Gly Ala        115           #       120           #       125Thr Gly Asp Phe Ala Gly Ser Asn Ser Gln Me #t Ala Gln Val Gly Asp    130               #   135               #   140Gly Asp Asn Ser Pro Leu Met Asn Asn Phe Ar #g Gln Tyr Leu Pro Ser145                 1 #50                 1 #55                 1 #60Leu Pro Gln Ser Val Glu Cys Arg Pro Phe Va #l Phe Gly Ala Gly Lys                165   #               170   #               175Pro Tyr Glu Phe Ser Ile Asp Cys Asp Lys Il #e Asn Leu Phe Arg Gly            180       #           185       #           190Val Phe Ala Phe Leu Leu Tyr Val Ala Thr Ph #e Met Tyr Val Phe Ser        195           #       200           #       205Thr Phe Ala Asn Ile Leu Arg Asn Lys Glu Se #r     210              #   215 <210> SEQ ID NO 28 <211> LENGTH: 65 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 28Met Lys Lys Thr Ala Ile Ala Ile Ala Val Al #a Leu Ala Gly Phe Ala1               5    #                10   #                15Thr Val Ala Gln Ala Asp Tyr Cys Asp Ile Gl #u Phe Gly Gly Gly Gly            20       #            25       #            30Ser Met Ser Val Leu Val Tyr Ser Phe Ala Se #r Phe Val Leu Gly Trp        35           #        40           #        45Cys Leu Arg Ser Gly Ile Thr Tyr Phe Thr Ar #g Leu Met Glu Thr Ser    50               #    55               #    60 Ser 65<210> SEQ ID NO 29 <211> LENGTH: 16 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 29Ser Pro Gly Gly Ser Gly Gly Ala Pro His Hi #s His His His His Cys1               5    #                10   #                15<210> SEQ ID NO 30 <211> LENGTH: 21 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 30Glu Phe Asp Tyr Lys Asp Asp Asp Asp Lys Gl #y Ala Pro Trp Ser His1               5    #                10   #                15Pro Gln Phe Glu Lys             20 <210> SEQ ID NO 31 <211> LENGTH: 24<212> TYPE: PRT <213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 31Glu Phe Glu Gln Lys Leu Ile Ser Glu Glu As #p Leu Asn Gly Ala Pro1               5    #                10   #                15Trp Ser His Pro Gln Phe Glu Lys             20 <210> SEQ ID NO 32<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: synthetic       module <400> SEQUENCE: 32Glu Phe Pro Gly Gly Ser Gly Gly Ala Pro Hi #s His His His His His1               5    #                10   #                15 Cys<210> SEQ ID NO 33 <211> LENGTH: 22 <212> TYPE: PRT<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: synthetic      module <400> SEQUENCE: 33Cys Glu Phe Asp Tyr Lys Asp Asp Asp Asp Ly #s Gly Ala Pro Trp Ser1               5    #                10   #                15His Pro Gln Phe Glu Lys             20 <210> SEQ ID NO 34<211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: synthetic       module <400> SEQUENCE: 34Cys Glu Phe Glu Gln Lys Leu Ile Ser Glu Gl #u Asp Leu Asn Gly Ala1               5    #                10   #                15Pro Trp Ser His Pro Gln Phe Glu Lys             20       #            25<210> SEQ ID NO 35 <211> LENGTH: 4380 <212> TYPE: DNA<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: vector<400> SEQUENCE: 35tctagagcat gcgtaggaga aaataaaatg aaacaaagca ctattgcact gg#cactctta     60ccgttgctct tcacccctgt taccaaagcc gactacaaag atgaagtgca at#tggtggaa    120agcggcggcg gcctggtgca accgggcggc agcctgcgtc tgagctgcgc gg#cctccgga    180tttaccttta gcagctatgc gatgagctgg gtgcgccaag cccctgggaa gg#gtctcgag    240tgggtgagcg cgattagcgg tagcggcggc agcacctatt atgcggatag cg#tgaaaggc    300cgttttacca tttcacgtga taattcgaaa aacaccctgt atctgcaaat ga#acagcctg    360cgtgcggaag atacggccgt gtattattgc gcgcgtcgtt ctggtgctta tg#attattgg    420ggccaaggca ccctggtgac ggttagctca gcgggtggcg gttctggcgg cg#gtgggagc    480ggtggcggtg gttctggcgg tggtggttcc gatatcgtga tgacccagag cc#cggatagc    540ctggcggtga gcctgggcga acgtgcgacc attaactgca gaagcagcca ga#gcgtgctg    600tatagcagca acaacaaaaa ctatctggcg tggtaccagc agaaaccagg tc#agccgccg    660aaactattaa tttattgggc atccacccgt gaaagcgggg tcccggatcg tt#ttagcggc    720tctggatccg gcactgattt taccctgacc atttcgtccc tgcaagctga ag#acgtggcg    780gtgtattatt gccagcagta ttcttctttt cctcttacct ttggccaggg ta#cgaaagtt    840gaaattaaac gtacggaatt cccagggggg agcggaggcg cgccgcacca tc#atcaccat    900cactgataag cttgacctgt gaagtgaaaa atggcgcaga ttgtgcgaca tt#ttttttgt    960ctgccgttta attaaagggg ggggggggcc ggcctggggg ggggtgtaca tg#aaattgta   1020aacgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc at#tttttaac   1080caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga ga#tagggttg   1140agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc ca#acgtcaaa   1200gggcgaaaaa ccgtctatca gggcgatggc ccactacgag aaccatcacc ct#aatcaagt   1260tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cc#cccgattt   1320agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa ag#cgaaagga   1380gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac ca#cacccgcc   1440gcgcttaatg cgccgctaca gggcgcgtgc tagactagtg tttaaaccgg ac#cggggggg   1500ggcttaagtg ggctgcaaaa caaaacggcc tcctgtcagg aagccgcttt ta#tcgggtag   1560cctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcatcag tg#aatcggcc   1620aacgcgcggg gagaggcggt ttgcgtattg ggagccaggg tggtttttct tt#tcaccagt   1680gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg ca#gcaagcgg   1740tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggtcag cg#gcgggata   1800taacatgagc tgtcctcggt atcgtcgtat cccactaccg agatgtccgc ac#caacgcgc   1860agcccggact cggtaatggc acgcattgcg cccagcgcca tctgatcgtt gg#caaccagc   1920atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa ac#cggacatg   1980gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt ga#gatattta   2040tgccagccag ccagacgcag acgcgccgag acagaactta atgggccagc ta#acagcgcg   2100atttgctggt ggcccaatgc gaccagatgc tccacgccca gtcgcgtacc gt#cctcatgg   2160gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cg#ccggaaca   2220ttagtgcagg cagcttccac agcaatagca tcctggtcat ccagcggata gt#taataatc   2280agcccactga cacgttgcgc gagaagattg tgcaccgccg ctttacaggc tt#cgacgccg   2340cttcgttcta ccatcgacac gaccacgctg gcacccagtt gatcggcgcg ag#atttaatc   2400gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gc#caatcagc   2460aacgactgtt tgcccgccag ttgttgtgcc acgcggttag gaatgtaatt ca#gctccgcc   2520atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gt#tcaccacg   2580cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cg#ttactggt   2640ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat ac#cgcgaaag   2700gttttgcgcc attcgatgct agccatgtga gcaaaaggcc agcaaaaggc ca#ggaaccgt   2760aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gc#atcacaaa   2820aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata cc#aggcgttt   2880ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cg#gatacctg   2940tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg ta#ggtatctc   3000agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cg#ttcagccc   3060gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag ac#acgactta   3120tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt ag#gcggtgct   3180acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt at#ttggtatc   3240tgcgctctgc tgtagccagt taccttcgga aaaagagttg gtagctcttg at#ccggcaaa   3300caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gc#gcagaaaa   3360aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gt#ggaacgaa   3420aactcacgtt aagggatttt ggtcagatct agcaccaggc gtttaagggc ac#caataact   3480gccttaaaaa aattacgccc cgccctgcca ctcatcgcag tactgttgta at#tcattaag   3540cattctgccg acatggaagc catcacaaac ggcatgatga acctgaatcg cc#agcggcat   3600cagcaccttg tcgccttgcg tataatattt gcccatagtg aaaacggggg cg#aagaagtt   3660gtccatattg gctacgttta aatcaaaact ggtgaaactc acccagggat tg#gctgagac   3720gaaaaacata ttctcaataa accctttagg gaaataggcc aggttttcac cg#taacacgc   3780cacatcttgc gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt ca#ctccagag   3840cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa caagggtgaa ca#ctatccca   3900tatcaccagc tcaccgtctt tcattgccat acggaactcc gggtgagcat tc#atcaggcg   3960ggcaagaatg tgaataaagg ccggataaaa cttgtgctta tttttcttta cg#gtctttaa   4020aaaggccgta atatccagct gaacggtctg gttataggta cattgagcaa ct#gactgaaa   4080tgcctcaaaa tgttctttac gatgccattg ggatatatca acggtggtat at#ccagtgat   4140ttttttctcc attttagctt ccttagctcc tgaaaatctc gataactcaa aa#aatacgcc   4200cggtagtgat cttatttcat tatggtgaaa gttggaacct cacccgacgt ct#aatgtgag   4260ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gt#atgttgtg   4320tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg at#tacgaatt   4380 <210> SEQ ID NO 36 <211> LENGTH: 2839 <212> TYPE: DNA<213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: vector<400> SEQUENCE: 36acccgacacc atcgaaatta atacgactca ctatagggag accacaacgg tt#tcccgaat     60tgtgagcgga taacaataga aataattttg tttaacttta agaaggagat at#atccatgg    120ctgaaactgt tgaaagttgt ttagcaaaat cccatacaga aaattcattt ac#taacgtct    180ggaaagacga caaaacttta gatcgttacg ctaactatga gggctgtctg tg#gaatgcta    240caggcgttgt agtttgtact ggtgacgaaa ctcagtgtta cggtacatgg gt#tcctattg    300ggcttgctat ccctgaaaat gagggtggtg gctctgaggg tggcggttct cc#gtacgacg    360ttccagacta cgcttccctg cgttcccatc accatcacca tcactaagct tc#agtcccgg    420gcagtggatc cggctgctaa caaagcccga aaggaagctg agttggctgc tg#ccaccgct    480gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg tt#ttttgctg    540aaaggaggaa ctatatccgg atcgagatcc ccacgcgccc tgtagcggcg ca#ttaagcgc    600ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc ta#gcgcccgc    660tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gt#caagctct    720aaatcggggc atccctttag ggttccgatt tagtgcttta cggcacctcg ac#cccaaaaa    780acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tt#tttcgccc    840tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg ga#acaacact    900caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cg#gcctattg    960gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa ta#ttaacgtt   1020tacaatttca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gt#ttattttt   1080ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tg#cttcaata   1140atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta tt#cccttttt   1200tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag ta#aaagatgc   1260tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gc#ggtaagat   1320ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aa#gttctgct   1380atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gc#cgcataca   1440ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc tt#acggatgg   1500catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ct#gcggccaa   1560cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc ac#aacatggg   1620ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca ta#ccaaacga   1680cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac ta#ttaactgg   1740cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cg#gataaagt   1800tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg at#aaatctgg   1860agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gt#aagccctc   1920ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac ga#aatagaca   1980gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aa#gtttactc   2040atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct ag#gtgaagat   2100cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc ac#tgagcgtc   2160agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gc#gtaatctg   2220ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg at#caagagct   2280accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa at#actgtcct   2340tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ct#acatacct   2400cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gt#cttaccgg   2460gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cg#gggggttc   2520gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc ta#cagcgtga   2580gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cg#gtaagcgg   2640cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct gg#tatcttta   2700tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gc#tcgtcagg   2760ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tg#gccttttg   2820 ctggcctttt gctcacatg              #                  #                 283 #9 <210> SEQ ID NO 37 <211> LENGTH: 4045<212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: vector<400> SEQUENCE: 37agcttaatta gctgagcttg gactcctgtt gatagatcca gtaatgacct ca#gaactcca     60tctggatttg ttcagaacgc tcggttgccg ccgggcgttt tttattggtg ag#aatccaag    120ctagcttggc gagattttca ggagctaagg aagctaaaat ggagaaaaaa at#cactggat    180ataccaccgt tgatatatcc caatggcatc gtaaagaaca ttttgaggca tt#tcagtcag    240ttgctcaatg tacctataac cagaccgttc agctggatat tacggccttt tt#aaagaccg    300taaagaaaaa taagcacaag ttttatccgg cctttattca cattcttgcc cg#cctgatga    360atgctcatcc ggaatttcgt atggcaatga aagacggtga gctggtgata tg#ggatagtg    420ttcacccttg ttacaccgtt ttccatgagc aaactgaaac gttttcatcg ct#ctggagtg    480aataccacga cgatttccgg cagtttctac acatatattc gcaagatgtg gc#gtgttacg    540gtgaaaacct ggcctatttc cctaaagggt ttattgagaa tatgtttttc gt#ctcagcca    600atccctgggt gagtttcacc agttttgatt taaacgtggc caatatggac aa#cttcttcg    660cccccgtttt caccatgcat gggcaaatat tatacgcaag gcgacaaggt gc#tgatgccg    720ctggcgattc aggttcatca tgccgtctgt gatggcttcc atgtcggcag aa#tgcttaat    780gaattacaac agtactgcga tgagtggcag ggcggggcgt aattttttta ag#gcagttat    840tggtgccctt aaacgcctgg ggtaatgact ctctagcttg aggcatcaaa ta#aaacgaaa    900ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga ac#gctctcct    960gagtaggaca aatccgccgc tctagagctg cctcgcgcgt ttcggtgatg ac#ggtgaaaa   1020cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg at#gccgggag   1080cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg ca#gccatgac   1140ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc ag#agcagatt   1200gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag ga#gaaaatac   1260cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt ct#gtcggctg   1320cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga at#caggggat   1380aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg ta#aaaaggcc   1440gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aa#atcgacgc   1500tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tc#cccctgga   1560agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gt#ccgccttt   1620ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct ca#gttcggtg   1680taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cg#accgctgc   1740gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt at#cgccactg   1800gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc ta#cagagttc   1860ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ct#gcgctctg   1920ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa ac#aaaccacc   1980gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aa#aaggatct   2040caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aa#actcacgt   2100taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tt#taaattaa   2160aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga ca#gttaccaa   2220tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc ca#tagctgcc   2280tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg cc#ccagtgct   2340gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aa#accagcca   2400gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat cc#agtctatt   2460aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg ca#acgttgtt   2520gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc at#tcagctcc   2580ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa ag#cggttagc   2640tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc ac#tcatggtt   2700atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt tt#ctgtgact   2760ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag tt#gctcttgc   2820ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gc#tcatcatt   2880ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag at#ccagttcg   2940atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac ca#gcgtttct   3000gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc ga#cacggaaa   3060tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gg#gttattgt   3120ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg gg#ttccgcgc   3180acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat ga#cattaacc   3240tataaaaata ggcgtatcac gaggcccttt cgtcttcacc tcgagaaatc at#aaaaaatt   3300tatttgcttt gtgagcggat aacaattata atagattcaa ttgtgagcgg at#aacaattt   3360cacacagaat tcattaaaga ggagaaatta accatgagtg acattgcctt ct#tgattgat   3420ggctctggta gcatcatccc acatgacttt cggcggatga aggagtttgt ct#caactgtg   3480atggagcaat taaaaaagtc caaaaccttg ttctctttga tgcagtactc tg#aagaattc   3540cggattcact ttaccttcaa agagttccag aacaacccta acccaagatc ac#tggtgaag   3600ccaataacgc agctgcttgg gcggacacac acggccacgg gcatccgcaa ag#tggtacga   3660gagctgttta acatcaccaa cggagcccga aagaatgcct ttaagatcct ag#ttgtcatc   3720acggatggag aaaagtttgg cgatcccttg ggatatgagg atgtcatccc tg#aggcagac   3780agagagggag tcattcgcta cgtcattggg gtgggagatg ccttccgcag tg#agaaatcc   3840cgccaagagc ttaataccat cgcatccaag ccgcctcgtg atcacgtgtt cc#aggtgaat   3900aactttgagg ctctgaagac cattcagaac cagcttcggg agaagatctt tg#cgatcgag   3960ggtactcaga caggaagtag cagctccttt gagcatgaga tgtctcagga aa#tcgaaggt   4020 agacatcacc atcaccatca ctaga          #                   #             4045 <210> SEQ ID NO 38<211> LENGTH: 1574 <212> TYPE: DNA <213> ORGANISM: artificial sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: expression       cassette <400> SEQUENCE: 38gctagcctga ggccagtttg ctcaggctct ccccgtggag gtaataattg ct#cgaccgat     60aaaagcggct tcctgacagg aggccgtttt gttttgcagc ccacctcaac gc#aattaatg    120tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gc#tcgtatgt    180tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac ca#tgattacg    240aatttctaga taacgagggc aaaaaatgaa aaagacagct atcgcgattg ca#gtggcact    300ggctggtttc gctaccgtag cgcaggccga ctactgcgat atcgaattcg ca#gaaacagt    360tgaaagttgt ttagcaaaac cccatacaga aaattcattt actaacgtct gg#aaagacga    420caaaacttta gatcgttacg ctaactatga gggctgtctg tggaatgcta ca#ggcgttgt    480agtttgtact ggtgacgaaa ctcagtgtta cggtacatgg gttcctattg gg#cttgctat    540ccctgaaaat gagggtggtg gctctgaggg tggcggttct gagggtggcg gc#tctgaggg    600tggcggtact aaacctcctg agtacggtga tacacctatt ccgggctata ct#tatatcaa    660ccctctcgac ggcacttatc cgcctggtac tgagcaaaac cccgctaatc ct#aatccttc    720tcttgaggag tctcagcctc ttaatacttt catgtttcag aataataggt tc#cgaaatag    780gcagggggca ttaactgttt atacgggcac tgttactcaa ggcactgacc cc#gttaaaac    840ttattaccag tacactcctg tatcatcaaa agccatgtat gacgcttact gg#aacggtaa    900attcagagac tgcgctttcc attctggctt taatgaggat ccattcgttt gt#gaatatca    960aggccaatcg tctgacctgc ctcaacctcc tgtcaatgct ggcggcggct ct#ggtggtgg   1020ttctggtggc ggctctgagg gtggcggctc tgagggtggc ggttctgagg gt#ggcggctc   1080tgagggtggc ggttccggtg gcggctccgg ttccggtgat tttgattatg aa#aaaatggc   1140aaacgctaat aagggggcta tgaccgaaaa tgccgatgaa aacgcgctac ag#tctgacgc   1200taaaggcaaa cttgattctg tcgctactga ttacggtgct gctatcgatg gt#ttcattgg   1260tgacgtttcc ggccttgcta atggtaatgg tgctactggt gattttgctg gc#tctaattc   1320ccaaatggct caagtcggtg acggtgataa ttcaccttta atgaataatt tc#cgtcaata   1380tttaccttct ttgcctcagt cggttgaatg tcgcccttat gtctttggcg ct#ggtaaacc   1440atatgaattt tctattgatt gtgacaaaat aaacttattc cgtggtgtct tt#gcgtttct   1500tttatatgtt gccaccttta tgtatgtatt ttcgacgttt gctaacatac tg#cgtaataa   1560 ggagtcttaa gctt               #                  #                   #   1574 <210> SEQ ID NO 39 <211> LENGTH: 932<212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial #Sequence: expression       cassette <400> SEQUENCE: 39gctagcctga ggccagtttg ctcaggctct ccccgtggag gtaataattg ct#cgaccgat     60aaaagcggct tcctgacagg aggccgtttt gttttgcagc ccacctcaac gc#aattaatg    120tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gc#tcgtatgt    180tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac ca#tgattacg    240aatttctaga taacgagggc aaaaaatgaa aaagacagct atcgcgattg ca#gtggcact    300ggctggtttc gctaccgtag cgcaggccga ctactgcgat atcgaattca at#gctggcgg    360cggctctggt ggtggttctg gtggcggctc tgagggtggt ggctctgagg gt#ggcggttc    420tgagggtggc ggctctgagg gaggcggttc cggtggtggc tctggttccg gt#gattttga    480ttatgaaaag atggcaaacg ctaataaggg ggctatgacc gaaaatgccg at#gaaaacgc    540gctacagtct gacgctaaag gcaaacttga ttctgtcgct actgattacg gt#gctgctat    600cgatggtttc attggtgacg tttccggcct tgctaatggt aatggtgcta ct#ggtgattt    660tgctggctct aattcccaaa tggctcaagt cggtgacggt gataattcac ct#ttaatgaa    720taatttccgt caatatttac cttccctccc tcaatcggtt gaatgtcgcc ct#tttgtctt    780tggcgctggt aaaccatatg aattttctat tgattgtgac aaaataaact ta#ttccgtgg    840tgtctttgcg tttcttttat atgttgccac ctttatgtat gtattttcta cg#tttgctaa    900 catactgcgt aataaggagt cttgataagc tt       #                   #         932 <210> SEQ ID NO 40 <211> LENGTH: 4425<212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: vector<400> SEQUENCE: 40tctagataac gagggcaaaa aatgaaaaag acagctatcg cgattgcagt gg#cactggct     60ggtttcgcta ccgtagcgca ggccgactac tgcgatatcg aattcgcaga aa#cagttgaa    120agttgtttag caaaacccca tacagaaaat tcatttacta acgtctggaa ag#acgacaaa    180actttagatc gttacgctaa ctatgagggc tgtctgtgga atgctacagg cg#ttgtagtt    240tgtactggtg acgaaactca gtgttacggt acatgggttc ctattgggct tg#ctatccct    300gaaaatgagg gtggtggctc tgagggtggc ggttctgagg gtggcggctc tg#agggtggc    360ggtactaaac ctcctgagta cggtgataca cctattccgg gctatactta ta#tcaaccct    420ctcgacggca cttatccgcc tggtactgag caaaaccccg ctaatcctaa tc#cttctctt    480gaggagtctc agcctcttaa tactttcatg tttcagaata ataggttccg aa#ataggcag    540ggggcattaa ctgtttatac gggcactgtt actcaaggca ctgaccccgt ta#aaacttat    600taccagtaca ctcctgtatc atcaaaagcc atgtatgacg cttactggaa cg#gtaaattc    660agagactgcg ctttccattc tggctttaat gaggatccat tcgtttgtga at#atcaaggc    720caatcgtctg acctgcctca acctcctgtc aatgctggcg gcggctctgg tg#gtggttct    780ggtggcggct ctgagggtgg cggctctgag ggtggcggtt ctgagggtgg cg#gctctgag    840ggtggcggtt ccggtggcgg ctccggttcc ggtgattttg attatgaaaa aa#tggcaaac    900gctaataagg gggctatgac cgaaaatgcc gatgaaaacg cgctacagtc tg#acgctaaa    960ggcaaacttg attctgtcgc tactgattac ggtgctgcta tcgatggttt ca#ttggtgac   1020gtttccggcc ttgctaatgg taatggtgct actggtgatt ttgctggctc ta#attcccaa   1080atggctcaag tcggtgacgg tgataattca cctttaatga ataatttccg tc#aatattta   1140ccttctttgc ctcagtcggt tgaatgtcgc ccttatgtct ttggcgctgg ta#aaccatat   1200gaattttcta ttgattgtga caaaataaac ttattccgtg gtgtctttgc gt#ttctttta   1260tatgttgcca cctttatgta tgtattttcg acgtttgcta acatactgcg ta#ataaggag   1320tcttaaggcc tgataagcat gcgtaggaga aaataaaatg aaacaaagca ct#attgcact   1380ggcactctta ccgttgctct tcacccctgt taccaaagcc gactacaaag at#gaagtgca   1440attggtggaa agcggcggcg gcctggtgca accgggcggc agcctgcgtc tg#agctgcgc   1500ggcctccgga tttaccttta gcagctatgc gatgagctgg gtgcgccaag cc#cctgggaa   1560gggtctcgag tgggtgagcg cgattagcgg tagcggcggc agcacctatt at#gcggatag   1620cgtgaaaggc cgttttacca tttcacgtga taattcgaaa aacaccctgt at#ctgcaaat   1680gaacagcctg cgtgcggaag atacggccgt gtattattgc gcgcgtcgtt ct#ggtgctta   1740tgattattgg ggccaaggca ccctggtgac ggttagctca gcgggtggcg gt#tctggcgg   1800cggtgggagc ggtggcggtg gttctggcgg tggtggttcc gatatcgtga tg#acccagag   1860cccggatagc ctggcggtga gcctgggcga acgtgcgacc attaactgca ga#agcagcca   1920gagcgtgctg tatagcagca acaacaaaaa ctatctggcg tggtaccagc ag#aaaccagg   1980tcagccgccg aaactattaa tttattgggc atccacccgt gaaagcgggg tc#ccggatcg   2040ttttagcggc tctggatccg gcactgattt taccctgacc atttcgtccc tg#caagctga   2100agacgtggcg gtgtattatt gccagcagta ttcttctttt cctcttacct tt#ggccaggg   2160tacgaaagtt gaaattaaac gtacggaatt cccagggggg agcggaggcg cg#ccgcacca   2220tcatcaccat cactgctgat aagcttgacc tgtgaagtga aaaatggcgc ag#attgtgcg   2280acattttttt tgtctgccgt ttaatgaaat tgtaaacgtt aatattttgt ta#aaattcgc   2340gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gc#aaaatccc   2400ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt gg#aacaagag   2460tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct at#cagggcga   2520tggcccacta cgagaaccat caccctaatc aagttttttg gggtcgaggt gc#cgtaaagc   2580actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa ag#ccggcgaa   2640cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tg#gcaagtgt   2700agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc ta#cagggcgc   2760gtgctagcca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa gg#ccgcgttg   2820ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg ac#gctcaagt   2880cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tg#gaagctcc   2940ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ct#ttctccct   3000tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc gg#tgtaggtc   3060gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agtccgaccg ct#gcgcctta   3120tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc ac#tggcagca   3180gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gt#tcttgaag   3240tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tc#tgctgtag   3300ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac ca#ccgctggt   3360agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg at#ctcaagaa   3420gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc ac#gttaaggg   3480attttggtca gatctagcac caggcgttta agggcaccaa taactgcctt aa#aaaaatta   3540cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcattc tg#ccgacatg   3600gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca cc#ttgtcgcc   3660ttgcgtataa tatttgccca tagtgaaaac gggggcgaag aagttgtcca ta#ttggctac   3720gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa ac#atattctc   3780aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat ct#tgcgaata   3840tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg aa#aacgtttc   3900agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca cc#agctcacc   3960gtctttcatt gccatacgga actccgggtg agcattcatc aggcgggcaa ga#atgtgaat   4020aaaggccgga taaaacttgt gcttattttt ctttacggtc tttaaaaagg cc#gtaatatc   4080cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct ca#aaatgttc   4140tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt tc#tccatttt   4200agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta gt#gatcttat   4260ttcattatgg tgaaagttgg aacctcaccc gacgtctaat gtgagttagc tc#actcatta   4320ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa tt#gtgagcgg   4380 ataacaattt cacacaggaa acagctatga ccatgattac gaatt   #                4425 <210> SEQ ID NO 41 <211> LENGTH: 5079<212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: vector<400> SEQUENCE: 41tctagataac gagggcaaaa aatgaaaaag acagctatcg cgattgcagt gg#cactggct     60ggtttcgcta ccgtagcgca ggccgatatc gtgctgaccc agccgccttc ag#tgagtggc    120gcaccaggtc agcgtgtgac catctcgtgt agcggcagca gcagcaacat tg#gcagcaac    180tatgtgagct ggtaccagca gttgcccggg acggcgccga aactgctgat tt#atgataac    240aaccagcgtc cctcaggcgt gccggatcgt tttagcggat ccaaaagcgg ca#ccagcgcg    300agccttgcga ttacgggcct gcaaagcgaa gacgaagcgg attattattg cc#agagctat    360gaccagaatg ctcttgttga ggtgtttggc ggcggcacga agttaaccgt tc#ttggccag    420ccgaaagccg caccgagtgt gacgctgttt ccgccgagca gcgaagaatt gc#aggcgaac    480aaagcgaccc tggtgtgcct gattagcgac ttttatccgg gagccgtgac ag#tggcctgg    540aaggcagata gcagccccgt caaggcggga gtggagacca ccacaccctc ca#aacaaagc    600aacaacaagt acgcggccag cagctatctg agcctgacgc ctgagcagtg ga#agtcccac    660agaagctaca gctgccaggt cacgcatgag gggagcaccg tggaaaaaac cg#ttgcgccg    720actgaggcct ctccaggggg gagcggaggc gcgccgcacc atcatcacca tc#actgctga    780taatatgcat gcgtaggaga aaataaaatg aaacaaagca ctattgcact gg#cactctta    840ccgttgctct tcacccctgt taccaaagcc caggtgcaat tgaaagaaag cg#gcccggcc    900ctggtgaaac cgacccaaac cctgaccctg acctgtacct tttccggatt ta#gcctgtcc    960acgtctggcg ttggcgtggg ctggattcgc cagccgcctg ggaaagccct cg#agtggctg   1020gctctgattg attgggatga tgataagtat tatagcacca gcctgaaaac gc#gtctgacc   1080attagcaaag atacttcgaa aaatcaggtg gtgctgacta tgaccaacat gg#acccggtg   1140gatacggcca cctattattg cgcgcgtttt gatccttttt ttgattcttt tt#ttgattat   1200tggggccaag gcaccctggt gacggttagc tcagcgtcga ccaaaggtcc aa#gcgtgttt   1260ccgctggctc cgagcagcaa aagcaccagc ggcggcacgg ctgccctggg ct#gcctggtt   1320aaagattatt tcccggaacc agtcaccgtg agctggaaca gcggggcgct ga#ccagcggc   1380gtgcatacct ttccggcggt gctgcaaagc agcggcctgt atagcctgag ca#gcgttgtg   1440accgtgccga gcagcagctt aggcactcag acctatattt gcaacgtgaa cc#ataaaccg   1500agcaacacca aagtggataa aaaagtggaa ccgaaaagcg aattcgacta ta#aagatgac   1560gatgacaaag gcgcgccgtg gagccacccg cagtttgaaa aatgataagc tt#gacctgtg   1620aagtgaaaaa tggcgcagat tgtgcgacat tttttttgtc tgccgtttaa tt#aaaggggg   1680gggggggccg gcctgggggg gggtgtacat gaaattgtaa acgttaatat tt#tgttaaaa   1740ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aa#tcggcaaa   1800atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc ag#tttggaac   1860aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cg#tctatcag   1920ggcgatggcc cactacgaga accatcaccc taatcaagtt ttttggggtc ga#ggtgccgt   1980aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gg#gaaagccg   2040gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag gg#cgctggca   2100agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gc#cgctacag   2160ggcgcgtgct agactagtgt ttaaaccgga ccgggggggg gcttaagtgg gc#tgcaaaac   2220aaaacggcct cctgtcagga agccgctttt atcgggtagc ctcactgccc gc#tttccagt   2280cgggaaacct gtcgtgccag ctgcatcagt gaatcggcca acgcgcgggg ag#aggcggtt   2340tgcgtattgg gagccagggt ggtttttctt ttcaccagtg agacgggcaa ca#gctgattg   2400cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt tt#gccccagc   2460aggcgaaaat cctgtttgat ggtggtcagc ggcgggatat aacatgagct gt#cctcggta   2520tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc gg#taatggca   2580cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aa#cgatgccc   2640tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gc#cttcccgt   2700tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc ca#gacgcaga   2760cgcgccgaga cagaacttaa tgggccagct aacagcgcga tttgctggtg gc#ccaatgcg   2820accagatgct ccacgcccag tcgcgtaccg tcctcatggg agaaaataat ac#tgttgatg   2880ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc ag#cttccaca   2940gcaatagcat cctggtcatc cagcggatag ttaataatca gcccactgac ac#gttgcgcg   3000agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac ca#tcgacacg   3060accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat tt#gcgacggc   3120gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gc#ccgccagt   3180tgttgtgcca cgcggttagg aatgtaattc agctccgcca tcgccgcttc ca#ctttttcc   3240cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ct#gataagag   3300acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac ca#ccctgaat   3360tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca tt#cgatgcta   3420gccatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gt#tgctggcg   3480tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aa#gtcagagg   3540tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ct#ccctcgtg   3600cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cc#cttcggga   3660agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta gg#tcgttcgc   3720tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc ct#tatccggt   3780aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc ag#cagccact   3840ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt ga#agtggtgg   3900cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gt#agccagtt   3960accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tg#gtagcggt   4020ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca ag#aagatcct   4080ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta ag#ggattttg   4140gtcagatcta gcaccaggcg tttaagggca ccaataactg ccttaaaaaa at#tacgcccc   4200gccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga ca#tggaagcc   4260atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cg#ccttgcgt   4320ataatatttg cccatagtga aaacgggggc gaagaagttg tccatattgg ct#acgtttaa   4380atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat tc#tcaataaa   4440ccctttaggg aaataggcca ggttttcacc gtaacacgcc acatcttgcg aa#tatatgtg   4500tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg tt#tcagtttg   4560ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct ca#ccgtcttt   4620cattgccata cggaactccg ggtgagcatt catcaggcgg gcaagaatgt ga#ataaaggc   4680cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa ta#tccagctg   4740aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat gt#tctttacg   4800atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca tt#ttagcttc   4860cttagctcct gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc tt#atttcatt   4920atggtgaaag ttggaacctc acccgacgtc taatgtgagt tagctcactc at#taggcacc   4980ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gc#ggataaca   5040 atttcacaca ggaaacagct atgaccatga ttacgaatt      #                   #  5079

What is claimed is:
 1. A method for displaying a (poly)peptide/proteinon the surface of a bacteriophage particle comprising: causing orallowing the attachment of said (poly)peptide/protein after itsexpression in an appropriate host cell to a member of the protein coatof said bacteriophage particle being assembled in said host cell,wherein said attachment is caused by the formation of a disulfide bondin said host cell between a first cysteine residue comprised in said(poly)peptide/protein and a second cysteine residue comprised in saidmember of the protein coat.
 2. The method of claim 1, wherein saidsecond cysteine residue is present at a corresponding amino acidposition in a wild type coat protein of a bacteriophage.
 3. The methodof claim 2, wherein said member of the protein coat is a wild type coatprotein of a bacteriophage.
 4. The method of claim 2, wherein saidmember of the protein coat is a truncated variant of a wild type coatprotein of a bacteriophage, wherein said truncated variant comprises atleast that part of said wild type coat protein causing the incorporationof said coat protein into the protein coat of the bacteriophageparticle.
 5. The method of claim 2, wherein said member of the proteincoat is a modified variant of a wild type coat protein of abacteriophage, wherein said modified variant is capable of beingincorporated into the protein coat of the bacteriophage particle.
 6. Themethod of claim 1, wherein said second cysteine residue is not presentat a corresponding amino acid position in a wild type coat protein of abacteriophage.
 7. The method of claim 6, wherein said second cysteinehas been artificially introduced into a wild type coat protein of abacteriophage.
 8. The method of claim 6, wherein said second cysteinehas been artificially introduced into a truncated variant of a wild typecoat protein of a bacteriophage.
 9. The method of claim 6, wherein saidsecond cysteine has been artificially introduced into a modified variantof a wild type coat protein of a bacteriophage.
 10. The method of anyone of claims 4 to 9, wherein said second cysteine is present at, or inthe vicinity of, the C- or the N-terminus of said member of the phagecoat of said bacteriophage particle.
 11. The method of claim 1, whereinsaid bacteriophage is a filamentous bacteriophage.
 12. The method ofclaim 11, wherein said member of the protein coat of the bacteriophageparticle is or is derived from the wild type coat protein pIII.
 13. Themethod of claim 11, wherein said member of the protein coat of thebacteriophage particle is or is derived from the wild type coat proteinpIX.
 14. The method of claim 1, comprising: (a) providing a host cellharbouring a nucleic acid sequence comprising a nucleic acid sequenceencoding said (poly)peptide/protein; (b) causing or allowing theexpression of said nucleic acid sequence; and (c) causing or allowingthe production of bacteriophage particles in said host cell.
 15. Themethod of claim 1, wherein said (poly)peptide/protein comprises animmunoglobulin or a functional fragment thereof.
 16. The method of claim15, wherein said functional fragment is an scFv or Fab fragment.
 17. Themethod of claim 1, wherein said (poly)peptide/protein is a member of adiverse collection of(poly)peptides/proteins displayed on a diversecollection of a plurality of bacteriophage particles.
 18. A method fordisplaying a (poly)peptide/protein on the surface of a bacteriophageparticle comprising the step of: causing or allowing the attachment ofsaid (poly)peptide/protein after its expression in an appropriate hostcell to a member of the protein coat of said bacteriophage particlebeing assembled in said host cell, wherein said attachment is caused bythe formation of a disulfide bond between a first cysteine residuecomprised in said (poly)peptide/protein and a second cysteine residuecomprised in said member of the protein coat, wherein said(poly)peptide/protein is a member of a diverse collection of(poly)peptides/proteins displayed on a diverse collection of a pluralityof bacteriophage particles.
 19. The method of claim 18, wherein saidbacteriophage is a filamentous bacteriophage.